WO2021243020A1 - Implantable device and method for in-situ detection of circulating cell-free tumor dna - Google Patents

Abstract

Disclosed herein are improvements to detection of circulating tumor DNA (ctDNA). An indwelling device may continually survey plasma volume (thus eliminating serial blood draws and repeat runs of sequencing and also obviating the problem of limited sampling volume in very low allele frequencies). Devices may employ, for example, DNA tweezers or other DNA probes / nano-devices, which may comprise a normal strand (N) with only natural DNA bases, and a complementary weak strand (W) containing one or more inosine (I) substitutions for guanine. An opening strand (O) or "invading strand," which is naturally complementary to N, may be ctDNA to be detected. The operation of the DNA probes may be driven by competition among N, W, and O strands. If O (invading strand) has sufficient affinity to forfeit N from W, the opening operation occurs and a resulting signal may be detected.

Description

IMPLANTABLE DEVICE AND METHOD FOR IN-SITU DETECTION OF CIRCULATING CELL-FREE TUMOR DNA

STATEMENT OF RELATED APPLICATIONS

[0001] This application claims the benefit of and priority as a PCT Application to U.S. Provisional Patent Application No. 63/038647, filed June 12, 2020, and U.S. Provisional Patent Application No. 63/031294, filed May 28, 2020, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] This disclosure relates generally to detecting circulating tumor DNA (ctDNA). An indwelling device may continually survey the plasma volume, and may employ, for example, DNA tweezers or other DNA probes / nano-devices that may be driven by competition among normal, weak, and opening strands that generate detectable signals.

BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Currently, patients are treated with post-operative (or “adjuvant”) chemotherapy and radiotherapy to address the risk of microscopic residual disease (i.e. remnant cancer cells). Using breast cancer as an example, even a margin-negative lumpectomy or mastectomy does not ensure comfortably low recurrence rates in the breast or elsewhere, thus leading to post operative chemotherapy and radiotherapy, based on probabilities of recurrence. Still, the majority of breast cancer patients have, in fact, been cured by surgery and microscopic residual disease remains only in a minority of patients, which demonstrates that there is significant exposure of patients to over treatment.

[0005] Accordingly, there is an urgent need for systems and methods that accurately monitor the risk of microscopic residual disease. SUMMARY

[0006] A fundamental problem in current cancer therapy is: how can we reliably identify the minority of patients who require post-operative chemo or RT, thus decreasing the massive overtreatment in breast, colorectal, lung cancers, and many other malignancies. The problem may be solved by non-invasive detection of circulating cell-free tumor DNA (ctDNA). In a liquid biopsy, blood is drawn, processed and then analyzed for the presence of ctDNA. However, current highly sensitive methods to detect ctDNA are unable to reliably detect ctDNA in the adjuvant (post-operative) setting, although the technology is successful in metastastic settings where there is abundant ctDNA. Disclosed are embodiments of an implantable device that monitors ctDNA in the plasma and electrically records signals when hybridization events have occurred, i.e. an implantable tracker of ctDNA. Embodiments of the disclosed approach address at least two problems, the limited sensitivity of detection with extra-corporeal ctDNA technologies when mutant allele frequencies are very low (i.e. atto to pico molar), and 2) the need for multiple serial collections to dynamically monitor the tumor state.

[0007] In a first aspect, various embodiments of the disclosure relate to a biodetector system, such as a vascular access port (VAP) system, comprising an implantable indwelling device for in-situ detection of nucleic acid sequences of interest, such as circulating cell-free tumor DNA (ctDNA) or other DNA. The indwelling device may comprise a catheter configured to interface with a circulatory system of a subject to access a bodily fluid in the circulatory system of the subject. The catheter may extend from a reservoir of the indwelling device. The indwelling device may comprise a DNA detector. The DNA detector may comprise a set of one or more DNA probes positioned at the catheter. The DNA probes may be, for example, on the catheter. The set of DNA probes may comprise one DNA probe or may comprise a plurality of DNA probes. One or more of the DNA probes in the set of DNA probes may comprise a normal DNA strand that is complementary to a ctDNA strand. For example, the set of DNA probes may comprise at least a first DNA probe with a first normal DNA strand complementary to a first ctDNA strand, and a second DNA probe with a second normal DNA strand complementary to the first ctDNA strand or to a second ctDNA strand. The DNA detector may comprise a set of one or more sensors configured to detect signals corresponding to DNA hybridizations at the one or more DNA probes. The set of sensors may comprise one sensor or may comprise a plurality of sensors. For example, the set of sensors may comprise at least a first sensor situated at the first DNA probe and configured to detect signals (e.g., electrical signals, optical signals, etc.) corresponding to hybridization of the first normal DNA strand to the first ctDNA strand, and a second sensor situated at the second DNA probe and configured to detect signals corresponding to hybridization of the second normal DNA strand to the second ctDNA strand. The proportion of sensors to DNA probes may vary. For example, the set of one or more sensors may comprise a sensor for each DNA probe (1 to 1 ratio) , a sensor corresponding to multiple DNA probes (1 to 2+ ratio), or multiple sensors corresponding to each DNA probe (2+ to 1 ratio).

[0008] In various embodiments, the normal strand of the at least one DNA probe may comprise a receptive region complementary to the ctDNA strand. For example, the first and second normal strands may comprise first and second receptive regions complementary to the first and second ctDNA strands, respectively. The first and second receptive regions may comprise DNA bases selected from a first group of DNA bases consisting of adenine (A), guanine (G), cytosine (C), and thymine (T).

[0009] In various embodiments, the at least one DNA probe may further comprise a weak strand complementary to the receptive region of the normal strand. For example, the first DNA probe may further comprise a first weak strand complementary to the first receptive region of the first normal strand. The second DNA probe may further comprise a second weak strand complementary to the second receptive region of the second normal strand. The one or more weak strands may comprise one or more inosine (I) substitutions for guanine (G). The first and second weak strands may comprise DNA bases selected from a second group of DNA bases consisting of adenine (A), guanine (G), inosine (I), cytosine (C) and thymine (T).

[0010] In various embodiments of the at least one DNA probe, the weak strand may be configured to decouple from the normal strand in the presence of the ctDNA strand. For example, the first weak strand may be configured to decouple from the first normal strand in the presence of the first ctDNA strand. The second weak strand may be configured to decouple from the second normal strand in the presence of the second ctDNA strand.

[0011] In various embodiments, the indwelling device may comprise at least one graphene surface to which the normal strand of the at least one DNA probe is secured. For example, the indwelling device may comprise a first graphene surface to which the first normal strand is secured. The indwelling device may comprise a second graphene surface to which the second normal strand is secured.

[0012] In various embodiments, the set of one more sensors may comprise at least one field effect transistor (FET) configured to detect electrical signals from hybridizations at the at least one DNA probe. For example, the first sensor may comprise a first FET configured to detect electrical signals from hybridizations at the first DNA probe. The second sensor may comprise a second FET configured to detect electrical signals from hybridizations at the second DNA probe.

[0013] In various embodiments, one or more of the at least one FET may be a graphene FET (GFET). For example, a subset of the FETs may be GFETs, or all of the FETs may be GFETs.

[0014] In various embodiments, the indwelling device may further comprise a microprocessor coupled, via electrodes, to source terminals and drain terminals of the at least one FET.

[0015] In various embodiments, the DNA detector may comprise a first graphene cuff. The DNA detector may additionally comprise a second graphene cuff. Each cuff may extend from a first end to a second end. The first and/or second graphene cuffs may encircle an external surface of the catheter. In some embodiments, the graphene cuffs may encircle an inside surface of the catheter. The first and second ends of the cuffs may be separated such that they do not make contact with each other circumferentially.

[0016] In various embodiments, first and second graphene cuffs may comprise the at least one DNA probe in varying proportions (e.g., one DNA probe per graphene cuff, two or more DNA probes per graphene cuff, 2 or more graphene cuffs per DNA probe, etc.). For example, the first graphene cuff may comprise the first DNA probe. The first graphene cuff may comprise the first sensor, which may comprise a first pair of electrodes at the first and second ends of the first graphene cuff. The second graphene cuff may comprise the second DNA probe. The second graphene cuff may comprise the second sensor, which may comprise a second pair of electrodes at the first and second ends of the second graphene cuff. [0017] In various embodiments, the indwelling device may further comprise a wireless transmitter configured to wirelessly transmit signals detected via the set of sensors to a remote computing device.

[0018] In various embodiments, the indwelling device may further comprise a thermal element coupled to a controller. The controller may be configured to use the thermal element to heat one or more DNA probes in the set of DNA probes.

[0019] In various embodiments, the controller may be configured to use the thermal element to heat one or more of the at least one DNA probe to a melting temperature at which the normal strand liberates the ctDNA strand. For example, the controller may be configured to use the thermal element to heat the first DNA probe and/or the second DNA probe to a melting temperature at which the first normal strand and/or the second normal strand liberates the first ctDNA strand and/or the second ctDNA strand, respectively.

[0020] In various embodiments, the indwelling device may further comprise a power supply comprising a battery and a charging module coupled thereto. The charging module may be an inductive charging module. The charging module may be situated at a diaphragm of the reservoir.

[0021] In various embodiments, the indwelling device may further comprise a wireless transmitter which may be configured to wirelessly transmit signals detected via the set of sensors to a computing device.

[0022] In various embodiments, the DNA detector may be configured to detect one or more variants identified in a tumor of the subject (such as a primary tumor surgically removed and sequenced to identify unique single-nucleotide variants (SNVs) that are present in the tumor but that are not present in germline control cells).

[0023] In various embodiments, the DNA detector may be configured to detect ctDNA in and/or on the catheter, and/or in and/or on the reservoir.

[0024] In another aspect, various embodiments may relate to a method of detecting circulating cell-free tumor DNA (ctDNA) in situ in a subject using a vascular access port system. The subject may be a post-operative cancer patient. The method may comprise implanting an indwelling device in the patient. The indwelling device may comprise a reservoir and a catheter extending from the reservoir. The catheter may be configured to interface, when implanted, with a circulatory system of the patient to access the patient’s blood. The indwelling device may comprise a DNA detector. The DNA detector may comprise a set of one or more DNA probes, which may be positioned at the catheter and/or the reservoir. The set of DNA probes may comprise one DNA probe or may comprise a plurality of DNA probes. One or more (or all) of the DNA probes in the set of DNA probes may comprise a normal DNA strand complementary to a ctDNA strand. For example, the set of DNA probes may comprise at least a first DNA probe. The first DNA probe may comprise a first normal DNA strand, which may be complementary to a first ctDNA strand. The first DNA probe may additionally comprise a second DNA probe with a second normal DNA strand, which may be complementary to the first ctDNA strand or to a second ctDNA strand. The indwelling device may comprise a set of one or more sensors configured to detect signals (e.g., electrical, optical, etc.) resulting from or otherwise corresponding to DNA hybridizations at one or more of the DNA probes. The set of sensors may comprise one sensor or may comprise a plurality of sensors. For example, the set of sensors may comprise at least a first sensor situated at the first DNA probe. The first sensor may be configured to detect signals corresponding to hybridization of the first normal DNA strand to the first ctDNA strand. The set of sensors may comprise a second sensor situated at the second DNA probe. The second sensor may be configured to detect signals corresponding to hybridization of the second normal DNA strand to the second ctDNA strand. The proportion of sensors to DNA probes may vary. For example, the set of one or more sensors may comprise a sensor for each DNA probe (1 to 1 ratio) , a sensor corresponding to multiple DNA probes (1 to 2+ ratio), or multiple sensors corresponding to each DNA probe (2+ to 1 ratio). The indwelling device may comprise a transmitter, such as a wireless transmitter. The transmitter may be configured to transmit detected signals and/or data based on detected signals. The wireless transmitter may be configured to wirelessly transmit to a remote computing device.

[0025] In various embodiments, the method may further comprise receiving signals and/or other data from the indwelling device. The signals may be received wirelessly. The signals may be received at the remote computing device. The method may comprise determining that ctDNA is present in the patient’s blood. For example, the method may comprise determining that the first ctDNA strand and/or the second ctDNA strand is present in the patient’s blood. The presence of the first and/or second ctDNA strands may be based on the signals received from the indwelling device.

[0026] In various embodiments, the indwelling device may further comprise a power supply. The power supply may comprise a battery. The power supply may additionally comprise a charging module coupled thereto. The charging module may be an inductive charging module configured to inductively receive energy with which the battery is charged. The charging module may be situated at a diaphragm of the reservoir. The method may further comprise inductively charging the battery of the indwelling device by positioning an inductive charger near the implanted reservoir of the indwelling device.

[0027] In various embodiments, the first DNA probe may further comprise a first weak strand complementary to a first receptive region of the first normal strand. The first weak strand may be configured to decouple from the first normal strand in the presence of the first ctDNA strand. The second DNA probe may further comprise a second weak strand complementary to a second receptive region of the second normal strand. The second weak strand may be configured to decouple from the second normal strand in the presence of the second ctDNA strand. The DNA detector may comprise a thermal element. The method may comprise heating, via the thermal element, the first and/or the second DNA probes to a melting temperature at which the first normal strand and/or the second normal strand liberates the first ctDNA strand and/or the second ctDNA strand, respectively.

[0028] In various embodiments, the method may comprise sequencing a tumor of the subject to identify one or more variants (e.g., SNVs) present in the tumor not present in germline control cells. The DNA detector may be configured to detect the one or more variants.

[0029] In various embodiments, the method may comprise surgically removing a primary tumor from the subject. The method may comprise sequencing the tumor to identify one or more SNVs present in the tumor. The method may comprise custom-designing one or more of the DNA probes in the DNA detector for detection of one or more of the identified SNVs.

[0030] Various embodiments provide an improvement in ctDNA detection technology wherein an indwelling device continually surveys the plasma volume (thus eliminating serial blood draws and repeat runs of sequencing and also obviating the problem of limited sampling volume in very low allele frequencies). Various embodiments may employ DNA tweezers, a DNA nano-device comprising three components: (i) a normal strand (N) containing only natural DNA bases [adenine (A), guanine (G), cytosine (C) and thymine (T)], (ii) a weak strand (W) containing one or more inosine (I) substitutions for guanine (with less energy than natural bonding to N), and (iii) the opening strand (O) or “invading strand,” which is naturally complementary to N. Inosine substitutions in W strands reduce the affinity of the W strand to the N strand, such that the W strand more “weakly” binds to the N strand relative to the O strand (which does not have inosine substitutions for guanine). In this case, ctDNA serves as the opening strand. The operation of the DNA tweezer is driven by competition among N, W and O. If O (or the invading strand) has even a single mismatch there is insufficient affinity to forfeit the binding of N to W, and the opening operation does not happen. If the invading strand has a sequence match with N, the tweezer strand is displaced towards the graphene surface. Because DNA is negatively-charged, this displacement, creates a detectable signal. Various embodiments employ the exonic SNVs, indels, and fusion sites observed in oncogenes for design of complementary weak (W) strands that are intentionally mismatched at the target loci (with our without additional guanine-inosine substitutions) to optimize the hybridization affinity of the complementary probes (bait or N strands) and weak strands such that ctDNA will efficiently displace the weak strand and create an electrical signal on a transistor. This signal can be wirelessly transmitted to computing devices for analysis, storage, and/or presentation.

[0031] In various embodiments, a microcircuit of graphene field effect transistors (FETs) may be used rather than a single FET with an array of DNA nanotweezers. Such complex patterning of graphene on a catheter may enable resolving which DNA nanotweezer generated the charge (e.g., whether it was p53 or BRAF-specific DNA nanotweezer).

[0032] In various embodiments, a device may be powered with a battery at the diaphragm that can be charged wirelessly. This may provide the minimal low voltage power needed for the processor(s) and importantly also allows for running of a reverse current back to the graphene FETs. A purpose of this is to heat the graphene (running the current over resistors for example at the metal graphene junctions) to the melting temperature of the duplexes.

This allows for periodic “resetting” of the device (liberating the captured strands, and reannealing of the nanotweezer). In various embodiments, a target of about 60 to 70 degrees may be employed at the graphene surface (from 40 degrees body temp). Graphene should heat quickly and dissipate quickly (e.g., a nanoampere (nA) current for a few seconds). [0033] In various embodiments, a DNA probe may comprise a DNA zipper that may be optimized to function in a flow environment. For example, a single stranded loop area of the DNA zipper may be moved to the top of the zipper region of the tweezer (i.e., more directly into the stream of flow), with all or most of the candidate sequence in the loop. This may be experimentally optimized. The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features will become apparent by reference to the following drawings and the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The foregoing and other objects, aspects, features, and advantages of the disclosure will become more apparent and better understood by referring to the following description taken in conjunction with the accompanying drawings, in which:

[0035] FIG. 1 illustrates a vascular access port system comprising an indwelling device and a computing device, according to various potential embodiments.

[0036] FIGs. 2A and 2B provide schematics of example DNA probes, illustrative of features of various potential embodiments.

[0037] FIGs. 3 A and 3B provide schematics of DNA nanosensors, illustrative of features of various potential embodiments.

[0038] FIG. 4 depicts an example implantable indwelling device, according to various potential embodiments.

[0039] FIGs. 5A and 5B provide a view of an example graphene grafted catheter, depicting a cartoon of a FET pattern running on the exterior surface of a patient catheter according to various potential embodiments. FIG. 5 A depicts an example indwelling device, and FIG. 5B depicts a cross section of a catheter of the device of FIG. 5 A, according to various potential embodiments.

[0040] FIG. 6 depicts a basic schematic of how multiple FETs may be hosted in a pattern on a YAP catheter according to various potential embodiments. [0041] FIG. 7 depicts a graphene monolayer adapted into a field-effect transistor with electrodes according to various potential embodiments.

[0042] FIG. 8 provides an example process for detecting sequences of nucleic acids of interest in a subject according to various potential embodiments.

[0043] FIG 9A: Cartoon reproduced from cancer.gov (accessed 6/6/20) demonstrating anatomic location and function of vascular access ports (VAPs). FIG. 9B: Illustrative schematic of graphene field effect transistor (FET) array with overlaying microfluidics chamber for in vitro testing and optimization of candidate nanotweezer probes, according to various potential embodiments. 9C: Illustrative cartoon of individual graphene FET with conjugated nanotweezer probe and invading strand (ctDNA) induced conformation change leading to triple-stranded DNA structure, according to various potential embodiments.

DETAILED DESCRIPTION

[0044] Various potential embodiments of the disclosed approach relate to an implantable device for the detection of circulating tumor DNA (ctDNA). DNA has a negative charge because of its sugar phosphate backbone and thus conformational changes can be detected through electrical methods. Bench-top technology for the electrical detection of specific hybridization events using novel DNA double-strand constructs referred to as DNA zippers or nanotweezers, combined with a label-free signal detection platform, may be used. Electronic detection platforms such as field-effect transistor (FET) sensors can be ideal - they are label-free and compatible with other electronic devices and are not expensive and do not require complex laser analyzers. Various embodiments of the disclosed approach may miniaturize and adapt this technology to host it on, for example, chemotherapy-delivery catheters for the detection of ctDNA, as a read out of residual tumor cells in the human body.

[0045] The disclosed approach has the potential to be transformative; the detection of post operative microscopic residual disease (or MRD) is one the “holy grails” of oncology. Even when tumors appear to be completely removed, the known risks of microscopic and undetectable cells remains too high in too many patients. Post-operative chemotherapy and radiation therapy is currently given empirically to all patients. However, if we could reliably and accurately identify patients who actually have microscopic residual disease, we could eliminate unnecessary chemotherapy and radiation treatments in hundreds of thousands of patients per year. Current approaches to this problem use bench-top next-generation sequencing (NGS) to look for ctDNA (a promising approach that is often described as “liquid biopsies”). Embodiments of the disclosed approach address key limitations of sequencing based approaches, and may include three components: 1) a totally-implantable vascular access port (VAP) that modifies existing patient devices but is engineered to “host” an array of FETs with oligonucleotide baits that can electrically detect and transmit hybridization signals; 2) a panel of tumor specific oligonucleotides probes (DNA nanotweezers) designed based on the recurrently mutated sequences in key oncogenes and/or tumor suprressors, and optimized constructs for hybridization efficiency in a vascular flow environment; 3) a device that incorporates these two components, which may be tested in simulated vascular flow experiments in vitro and then in large animal models.

[0046] Referring to FIG. 1, an example vascular access port system 100 comprises an indwelling device 110 and a computing device 162. The indwelling device 110 may comprise a DNA detector 114, an interface module 130, a power supply 146, and a transceiver 158. The DNA detector 114 may comprise one or more DNA probes 118, such as the probes illustrated in FIGs. 2A - 3B. In certain embodiments, different DNA probes 118 may be incorporated into one indwelling device 110, each probe configured to detect a different ctDNA strand. The DNA detector 114 may comprise one or more sensors 112, which may include one or more field effect transistors (FETs), such as graphene FETs that detect electrical signals from hybridization events at the DNA probes 114. In alternative embodiments, sensors may detect optical signals from, for example, fluorescently-labeled components of DNA probes 118. For example, a weak strand may be fluorescently labeled, and its displacement by a ctDNA strand may change impact optical readings (e.g., lower the intensity of light at the normal strand as detected using an optical sensor pointed at the normal strand) and indicate that a ctDNA strand has been detected. DNA detector 114 may comprise a thermal element 126, which may comprise one or more resistive elements that increase in temperature in response to a current being applied thereto. Thermal element 126 may be part of or in contact with DNA probes 118 to enable heating of portions of the DNA probes 118 to, for example, melting temperatures that decouple strands of DNA.

[0047] The interface module 130 of indwelling device 110 may comprise a controller 134, which may include a microprocessor 138. The controller 134 may include a memory 142 for storing detected signals and/or instructions executable by the microprocessor 138. The interface module 138 may serve as an interface between the DNA detector 114 and the power supply 146 and/or the transceiver 158. Microprocessor 137 may be coupled to the sensors 122 to receive signals detected from DNA probes 118, and to thermal element 126 to control application of a current to heat DNA probes 118 to “reset” DNA probes 118.

[0048] Power supply 146 of indwelling device 110 may comprise a battery 150, which may be rechargeable using a charging module 154. The charging module 154 may enable inductive charging of battery 150. A transceiver 158 may send and receive signals from computing device 158. In some embodiments, transceiver 158 may wirelessly send and/or receive signals through any suitable protocols that may employ, for example, radio frequency communications. In some embodiments, transceiver 158 is only transmitter for sending detected signals to computing device 162. In other embodiments, transceiver 158 enables indwelling device 110 to, for example, receive control signals, instructions, or other data from computing device 162. In some embodiments, transceiver 158 employs near field communications (NFC), requiring a computing device 162 to be brought, for example, within a few centimeters of indwelling device 110, or may employ protocols allowing for more distant communications, such as Wi-Fi or Bluetooth. In other embodiments, transceiver 158 may be used for wired communication with computing device 162.

[0049] Computing device 162 (or multiple computing devices, co-located or remote to each other) may include a controller 166, which may comprise a processor 168 and volatile and/or non-volatile memory 172, which may store, for example, instructions executable by the processor 168 in addition to software applications and signals received from indwelling device 110 and/or data based on such signals. The computing device 162 may include a user interface 176 that allows the computing device 162 to receive user inputs via input devices 180 (e.g., via a keyboard, touchscreen, microphone, camera, etc.) and provide outputs via output device 184 (e.g., via a display screen, audio speakers, etc.). A transceiver 188 allows the computing device 110 to receive and/or exchange readings, control commands, signals, and/or other data with indwelling device 110 wirelessly or via wires. The computing device 162 may additionally include one or more databases 192 for storing, for example, signals detected via one or more sensors 122 and/or interpretations of detected signals. In some implementations, database 192 (or portions thereof) may alternatively or additionally be part of another computing device that is co-located or remote and in communication with computing device 110 and/or indwelling device 110. [0050] FIGs. 2A and 2B illustrate example DNA probes according to various potential embodiments. In FIG. 2A, a double-stranded DNA probe (200) may comprise a normal strand (205) secured to a graphene surface (210) at a toehold region (215) of the normal strand (205). An opening strand (220), such as a circulating tumor DNA (ctDNA) strand to be detected, may displace a weak strand (225) that is complementary to a receptive region (230) of the normal strand (205). The opening strand (220) may have greater affinity for the normal strand (205) due to selection of DNA base pairs, as discussed below. Charges from ctDNA (220) accumulated at the toehold region (215) may be detected as nucleotides that are close to the graphene surface influence the electrostatic potential on a sensor and may generate a detectable electrical signal. In FIG. 2B, a DNA strand (250) that does not have greater affinity for the normal strand (205) than the weak strand (225) does not displace the weak strand (225) and thus does not generate a detectable signal.

[0051] FIGs. 3 A and 3B illustrate example DNA probes with DNA nanosensors according various potential embodiments. In FIG. 3 A, a DNA nanotweezers probe design is horizontally laid down on the graphene surface, placing a longer DNA sequence in a charge accumulation part (dotted circle) in close proximity to the graphene surface (longer than the few nucleotides at toehold 215 in FIG. 2A, thereby generating a stronger signal). When an opening strand (305) displaces the weak (“W”) strand from the normal (“N”) strand, the single-stranded toehold region becomes double-stranded, and the overall probe architecture becomes triple-stranded bringing the longer DNA sequences of the charge accumulation part closer to the surface than the double-stranded probe 205 and gives larger signal. However single-mismatch opening strand prohibits efficient strand displacement leaving the toehold region single-stranded. In FIG. 3B, schematics of graphene FET sensor with DNA tweezers probe are depicted. Gate voltage was applied directly on the liquid gate (shown as the hemisphere or bubble). I-V curve shifts leftward and downward only during the perfect match T strand displacement (inset). See Hwang, M. T. et al. DNA Nanotweezers and Graphene Transistor Enable Label-Free Genotyping. Adv Mater, el 802440, doi : 10.1002/adma.201802440 (2018).

[0052] Referring to FIG. 4, an example indwelling device 400 that may be implanted in a subject comprises a catheter 405 extending from a reservoir 410. The indwelling device 400 may contact blood or other bodily fluid while implanted in a subject. The reservoir 410 may comprise a diaphragm 420 through which, for example, biologically active compounds may be introduced to the subject. In various embodiments, one or more DNA detectors 425, 430, 435 may be positioned on the catheter and/or the reservoir. Other components may be situated at, for example, the reservoir at 440 (such as interface module 130, power supply 146, and/or transceiver 158), and may be connected to DNA detectors 425, 430, 435 via electrical pathways 445, 450, 455. In other embodiments, various components 440 may be integrated with DNA detectors 425, 430, 435 in or on the catheter 405 and/or in or on the reservoir 410. It is noted that, in various embodiments, the reservoir and catheter are not directly in plasma circulation. Detection may be occurring on the external surface of the catheter rather than the internal surface.

[0053] Referring to FIGs. 5A and 5B, an example indwelling device 500 comprises a catheter 505 extending from a port 510 (which may comprise a reservoir and diaphragm). The catheter 505 may comprise a set of graphene cuffs 520 situated along a length of catheter 505. Each graphene cuff 520 may comprise a DNA detector (comprising, e.g., DNA probes, electrical sensors, and/or thermal elements), such as a DNA probe secured to a graphene surface of a GFET. Referring to FIG. 6, an example catheter 605 with a set of graphene cuffs 630 is illustrated according to potential embodiments. The set of cuffs 620 may be connected to a microprocessor 630 via electrical pathways 630. Referring to FIG. 7, a DNA detector 705 may comprise a graphene surface 7210 on a silicon substrate 715. A DNA probe may be placed on the graphene surface 710 between a pair of electrodes 720 that may detect electrical signals due to hybridization events at the DNA probes.

[0054] Referring to FIG. 8, an example process 800 is depicted. At 810, an indwelling device may be implanted in a subject to detect nucleic acids with sequences of interest in a bodily fluid of the subject. The patient may be a post-operative cancer patient, and the indwelling device may be implanted to detect, for example, ctDNA in the patient’s blood.

The indwelling device may be or comprise a vascular access port with DNA detectors that include DNA probes for one or more gene regions. At 820, the indwelling device may be initiated for detection of signals corresponding to the presence of DNA of interest. Initiation may include powering on the indwelling device and/or confirming that a computing device may communicate with the indwelling device. In some embodiments, initiation may comprise using resetting the DNA probes by using a thermal element in the indwelling device to heat one or more DNA probes. At 830, signals may be received, wirelessly or using wires, from the indwelling device using a computing device. At 840, the signals may be used to determine, for example, that certain ctDNA is present in the blood of the subject. This may involve, for example, requiring a certain number of readings over a certain time so as to reduce false positives. At 850, the signals and/or interpretations thereof may be output on an output device of the computing device, and/or stored in a database of the computing device, for use in, for example, setting or adjusting clinical treatment protocols for the subject.

[0055] In a first aspect, various embodiments may comprise a totally implantable, intravascular venous access port (VAP) that preserves the current functions of infusion of chemotherapy agents but that also serves as a ctDNA biosensor. This may be achieved by modifying existing polyurethane and silicone VAPs (such as the popular “portacath”), where a microcircuit of graphene FETs may be fabricated on the external surface of the VAP. This microcircuit may host the electrical detection technology for the DNA hybridization technology in the second aspect (discussed below). As the DNA tweezer baits change configuration upon hybridization to ctDNA, they will modulate the current on the graphene FETs, which can be detected and then transmitted to extra-corporeal computing devices. Heating the FETs will liberate the candidate and reanneal (“re-zip”) the tweezer. VAPs currently in use may be modifiable with a patterned graphene FET microcircuit that 1) serves as an electrical detection system, and 2) can run a current back to the FET surfaces to achieve the modest heating needed to achieve DNA melting temperatures.

[0056] In a second aspect, a primary tumor may be sequenced. For example, the primary breast cancer or colon cancer (taken at surgery) may be sequenced. Once key discriminating mutations are known in the primary tumor, candidate probes may be designed for loading onto a catheter. In some embodiments, patient derived plasma can be deep sequenced using NGS technologies for candidate genomic targets that are recurrently mutated in cancers. These candidate sequences may be used to design novel DNA double strand nanotweezer constructs in which invasion and annealing by a complementary strand displaces the weak strand. Analytic performance of embodiments of the DNA double-stranded tweezer probes may be validated for detection of candidate ctDNA sequences in flow environments using standard in vitro fluorescence-based DNA strand displacement techniques and analyses. Notably, the probes can be optimized by modifying the number and location of inosine-for- guanine substitutions and the length or location of the toehold or loop regions. DNA tweezer probes may be optimizable for successful hybridization events in conditions that mimic central venous flow conditions. The sensing strand of tweezer strands can rehybridize (“re zip”) for subsequent events.

[0057] In various embodiments, candidate probes may be custom designed based on sequencing of a given patient’s primary tumor. For example, a woman may have a lumpectomy. The breast tumor that is removed may have NGS and unique SNVs (single nucleotide variants) may be identified in the tumor that are not present in sequencing of germline control cells. Then, custom probes may be designed for those SNV and customized patient specific, tumor specific catheters may be deployed.

[0058] In a third aspect, candidate double-strand DNA tweezer probes may be conjugated to the graphene FETs on the surface of prototype VAPs and evaluated for performance in an in vitro fluidics system that mimics vascular flow conditions. Synthesized ctDNA oligonucleotides may be added to the system to evaluate electrical signal detection performance of prototype devices. Once performance is satisfactory, the device may be deployed in a large animal model such as a pig model and will measure the sensitivity and specificity of the device based on known ctDNA fragment infusion into the peripheral venous circulation. The prototype may successfully trigger electrical signals in response to ctDNA <0.01% of total cell-free DNA.

[0059] With respect to the current landscape of ctDNA as a read out for minimum residual disease (or MRD), cell-free DNA (cfDNA) can exist in different states, including within microvesicles, or part of nucleosomes, or bound to plasma proteins¹. Most cfDNA is believed to be originate in passive shedding of DNA after cell death in normal processes. As such, most cfDNA comes from hematopoetic cells, which have high turn-over and high population numbers. Several inflammatory or traumatic triggers have been associated with higher levels of cfDNA. cfDNA typically is 150-200 bp, corresponding to the length of DNA for one nucleosome revolution (147bp) plus linker DNA associated with histone HI. Circulating tumor DNA (ctDNA) is one component of cfDNA and typically consists of shorter fragments of 90-150 bp^{2 4}. Detecting ctDNA is easier when it is relatively abundant, for example in metastatic states, when mutant allele fractions can be as high 1-10% of total cfDNA. In locally advanced, non-metastatic disease this frequency falls to under 1%. In early-stage disease (and after curative treatment) the frequency is usually under 0.1%⁵.

Hence, detection of these low frequencies poses a tractable technological problem. The problem is complicated however by the presence of benign variants, often due to clonal hematopoiesis in which age-associated acquired mutations can lead to false positives⁶. Current approaches to detecting these low level ctDNAs include extracorporeal in vitro techniques based either on PCR or on next-generation sequencing technologies (NGS). The earliest reported PCR method was amplification refractory mutation system or ARMS, in which PCR primers corresponding to polymorphic genomic candidates known to be mutated in malignancy were used. In this way, PCR amplification is conditional on a perfect hybridization of the primer with candidate mutations^7,8. However, only one locus can be queried per assay, thus limiting the overall utility. Digital PCR (dPCR) and digital droplet PCR (ddPCR) create in-parallel PCR reactions within aqueous droplets in an oil emulsion, with each droplet partitioning and amplifying a single DNA candidate^9,10. This approach has improved the detection rate but requires a priori knowledge of candidate mutations.

[0060] NGS based methods rely on targeted approaches that seek to strike a balance between sequencing depth and the cost of sequencing. In multiplex PCR-based NGS, PCR amplification of selected genes is accompanied by unique molecular identifier based multiplexing followed by sequencing¹¹. These methods can correct for amplification-based artifacts and can be very sensitive and specific, and also allow for personalization for known SNVs from the index tumor. Alternatively, in hybrid-capture based NGS approaches, recurrently mutated ctDNA candidate are “pulled-down” using biotinylated complementary probes, followed by sequencing¹². The candidates are selected using bioinformatic analyses of known datasets. Candidate SNVs, indels, and rearrangement sites can be detected with high accuracy. Finally, combination approaches using different (complementary) modalities combined with powerful computational techniques can lead to detection of minimum or microscopic residual disease (MRD), emerging clones, and phylogenetic relationships between disease sites.

[0061] Significance of various potential embodiments: The detection MRD has long been a “holy grail” in oncology. The entire premise of adjuvant therapy is based on the probability of microscopic MRD that can lead to relapse. Thus, all patients are treated based on observed relapse rates in clinical trials of adjuvantly treated patients vs not. However, with a reliable read-out of MRD, the premise of adjuvant therapy would dramatically change, focusing instead only patients with demonstrable MRD (rather than all patients). ctDNA holds promise as a read out for MRD but is currently limited by the threshold of detection for MRD, the need for serial blood draws for extracorporeal in vitro assays as described above, and the cost of sequencing. Still, current data are reason for optimism despite these limitations. Chaudhuri et al. demonstrated that ctDNA detection using CAPP-Seq at 4 months after curative treatment for NSCLC could predict the risk of relapse with 94% sensitivity and 100% specificity⁵. Similarly, Abbosh et al. demonstrated the feasibility of detecting MRD after curative treatment for NSCLC using a multiplexed PCR approach, including in patients with MAF of <0.1%^{13 14}. Turner et al. used a ddPCR approach in breast cancer, demonstrating that serial ctDNA collection could identify patients at higher risk of relapse, and could do so 8 months before clinical evidence of disease¹⁵. Similar work has been reported in colon cancer and other malignancies¹. We posit that a clear improvement in ctDNA detection technology would continually survey the plasma volume (thus eliminating serial blood draws and repeat runs of sequencing, as well as obviating the problem of limited sampling volume in very low allele frequencies). Such a technology would dramatically reduce overtreatment in hundreds of thousands of patients.

[0062] DNA nanosensor technologies: DNA strand displacement has been the central technique for DNA nano-manipulation, and occurs when a DNA double helix exchanges one strand for another complementary strand¹⁶. The introduced strand holds higher affinity to one strand in the initial double helix and displaces another strand. Inosine or RNA can be used to control hybridization kinetics or Gibbs free energy^{17 19}. Strand displacement-based assays can discriminate variants efficiently by controlling competition between initially hybridized parts in the double stranded or hairpin-structured probe and probe-to-target hybridization²⁰. These systems typically have fluorescence-based readouts. However, fluorescence-based sensors have limited lifetimes and background issues. Fluorescence-based detection also requires fluorimeters or laser scanners to analyze the optical signal. Molecular beacon probes are an example of fluorescence based nano-sensing of DNA^{20 22}.

[0063] A DNA nano-device called a DNA zipper provides the physical movement of DNA segments by alternating each other (FIGs. 2A, 2B, 3A, 3B)²³. Specifically, a DNA zipper contains three components: (i) a normal strand (N) containing only natural DNA bases [adenine (A), guanine (G), cytosine (C) and thymine (T)], (ii) a weak strand (W) containing inosine (I) substituted for guanine (with less energy than natural bonding to N), and (iii) the opening strand (O), which is naturally complementary to N. N or W can have a short length of toehold to facilitate the reaction. W and N are weakly bonded compared to N and O therefore introducing O allows the zipper to open. Using the DNA zipper as a probe enables distinguishing perfect matches from mismatched strands by its operation²⁴. There is not a single exposed strand on the probe thus it rules out the interference problem inherent with other molecular beacon probes. Controlling the energy distribution over the DNA zipper part, longer probes can also be designed. In a further improvement, the DNA probe can be laid horizontally on the detector surface (now referred to as DNA nano-tweezers) ²⁶. In this construct, the invading strand and hybridization does not displace the hinge component, thus resulting in a triple-strand complex, which can be electrically detected. DNA has negative charge due to its sugar phosphate backbone. Thus, the ionic charge difference between ssDNA and dsDNA is one fold. The difference between ssDNA and dsDNA can be measured if a field effect sensor is sensitive enough to detect a small charge of DNA²⁵, in this case the detection of a DNA hybridization event. Graphene provides a large detection area, biocompatibility, and exceptional electronic properties such as ultra-high mobility and ambipolar field-effect. The DNA nanotweezer can be functionalized on the surface of graphene and when an invading or opening strand O hybridizes the toehold part of N, which is adjacent to the graphene surface, becomes double-stranded after strand displacement creating an electrical signal that is demonstrable in an I-V curve. This type of hybridization event and the subsequent charge detection has been successfully tested ex vivo and reported in the Lai lab²⁶.

[0064] Innovations of various potential embodiments: Many biosensing applications in development rely on read-outs from chemistry reactions that are detected ex vivo and in rare instances, in vivo. Various embodiments of this application may take the ex vivo, bench-top demonstration of electrical detection of conformation-altering hybridization events and engineer them on to an implantable intravascular device that can continually monitor a patient for ctDNA. Using an implantable device to detect an electrical signal based on a DNA hybridization may be unprecedented as an application for detecting nucleic acids in the human body. As we discuss above, embodiments of the disclosed approach may significantly alter the landscape of post-operative cancer treatment over a relatively short timeline.

Various embodiments may comprise an implantable device that is an important screening tool that can be followed by NGS based methods for ctDNA detection of MRD in patients who are flagged as having detectable ctDNA. Various embodiments may modify devices that are already commonly used in cancer patients, and improve them with disclosed detection / sensor function as described below.

[0065] Example approach to the first aspect: an implantable functional venous access port that serves as a graphene based electrical transistor (see, e.g., FIGs. 5 - 7).

[0066] In various embodiments of this application, an indwelling device must have access to the plasma volume and thus has to be an intravascular device. In-dwelling central venous access ports with puncturable diaphragms may be used to deliver chemotherapy agents and for serial blood draws (commonly known as “porta-caths”), with access to the superior vena cava. The catheter may be configured to detect ctDNA as described below with respect to the second aspect, thus serving as a biosensor which transmits electrical signals to an external device. The device’s primary function may remain intact and could still be used to deliver systemic chemotherapies through the lumen. In an alternate use-case, an implantable device may in various embodiments be designed using the principles described below for use in craniospinal fluid of the brain, where ctDNA from brain tumors can be detected.

[0067] Currently available chemotherapy VAPs are 55-76 cm long and available in outer diameters of 1.9-3.2 mm, with the common 8 F devices having an OD of 2.6 -2.7 mm size. The catheters can be polyurethane (PU) or silicone. Various embodiments may modify the outer surface of the distal 10 cm of catheter length (representing the subclavian to superior vena cava length) with a fabricated microcircuit of graphene field-effect transistors (FETs) on the external surface of the VAP. This microcircuit may function as an electronics-based sensor for unique DNA hybridization events without the need for fluorescence labeling. Various embodiments may use an offset of 2 cm from the tip of the catheter to account for manufacturer specific tip improvements and to minimize the chance of retrograde flow of chemotherapy agents over the device.

[0068] Resolving the location of hybridization events to understand genomic sequences corresponding to ctDNA: various embodiments may employ a pattern of graphene islands (individual FETs) created on the external device that will allow us to “sectorize” the catheter surface and resolve the location of unique hybridization events as they occur so we can understand which genomic probe (see second aspect) has triggered. In possible embodiments, there will be 10 or more such graphene FETs on the catheter surface, each measuring 7 mm x 1-2 mm, and arranged along the length of the catheter. Each FET may be a unique electrode for charge-detection leading to one or more nanoscale microprocessors at the proximal end of the catheter at the level of the subcutaneous diaphragm. Each island may represent just one specific nanotweezer bait sequence (for example a specific hotspot mutation in the TP53 gene) or may host multiple baits. Probes placed in the same island/FET may be intentionally designed to create different charges. The genomic identity of each unique hybridization event may be resolved by 1) which FET the charge originated in, and 2) the size of the charge transfer.

[0069] Powerizing the device and resetting nanotweezers for future events by heating graphene islands in various embodiments: At the proximal end of the device (at the site of the puncturable diaphragm), the device may be re-engineered to host a battery. This battery may be remotely chargeable wirelessly by transferring energy from an external charging device with an induction coil to create an alternating electromagnetic field, with a receiver coil in the diaphragm that charges the battery. Low levels of power from the battery may be used to power the nanoscale processors on the device and for periodically running a current back to the graphene FET microcircuit (through a resistor) to heat the graphene regions to the melting temperature of double-stranded hybridization events. The melting temperature can be calculated by the length of the duplexed tweezer and the G-C content in the segment. The highest calculated melting temperature among the nanotweezer baits may be used as the reference target temperature. In various embodiments, this may potentially be approximately 60 70 degrees C (human body temperature is 40 degrees C). This will melt the invading strands off the device and will allow the nanotweezer to recouple its native dsDNA thus allowing for subsequent binding events. We anticipate this to be safe because graphene can rapidly transfer and dissipate heat. The charge will only run to the graphene island network and the heated volume may be small and super-local to the catheter surface. Various embodiments comprise a flat digital wireless chip with a patterned graphene FET microcircuit that corresponds to the dimensional surface available on VAPs. Amino modified DNA tweezers (see second aspect) may be conjugated on PBASE modified graphene microFETs and once we have demonstrated highly efficient and specific ctDNA detection on a flat circuit, we will adapt it to the catheter surface (see below).

[0070] Experimental methods with respect to various potential embodiments

[0071] CAD (computer-aided design) may be used to create a patterned FET array and microcircuit consistent with the available surface dimension of YAP catheters, as well as a battery system at the diaphragm. A design may be fabricated using advanced transparent printed micro-electronics technique. In various embodiments, at first, graphene FET microcircuit may be printed on an ultrathin flat silicone rubber surface (substrate / membrane). With a flat, 2-dimensional microcircuit, we may transfer the technology to catheters by thermal pressing or simply pressing. Alternatively the graphene FET biosensor system may be directly printed on polyurethane or silicone catheter using printed electronics techniques. The signal analyzer (microprocessor) may be connected / positioned at the diaphragm area, near the battery system.

[0072] Materials: Graphene may be purchased from Graphenea or other reputable sources. The FRDM KL-25Z microcontroller board may be purchased from Freescale Semiconductor.

[0073] Fabrication of graphene FET islands on catheter surface: Based on the e-beam lithography technique, circuit and array of graphene FET of - 1-2 mm x 7 mm size may be created. To deposit the graphene on the target surface, PMMA (a surface protective polymer) may be spin-coated on the top (carbon) surface of graphene/copper substrate (to protect from damage) and copper may be etched by floating on 0.1 M of ammonium persulfate for about 5 hours and rinsed in deionized (DI) water overnight. PMMA supported graphene may be transferred onto silicon dioxide coated catheter followed by removal of the PMMA layer with acetone at 60 °C for 1 hour. The sample may be annealed at 200 °C for 2 hours under hydrogen/argon atmosphere. Silicone rubber may be applied to insulate source and drain electrodes from liquid and used as a solution reservoir. FET biosensor microcircuit may be created for each installed graphene electrode. Each installed graphene electrode may act as individual signal/charge receptor. Received signals (electrical charges) from each hybridization may be channeled through the FET biosensor printed micro-circuit (as electrical current) to the signal detector and analyzer (microprocessor/microelectronics).

[0074] This can be done in two potential ways:

[0075] The entire FET biosensor system may be printed on ultrathin polyurethane or silicone membrane layer using a printed electronics technique and then attached to the catheter using heat press.

[0076] Alternatively the graphene FET biosensor system may be directly printed on polyurethane or silicone catheter using printed electronics techniques. [0077] The signal analyzer (microprocessor) may be connected / positioned at the diaphragm area, near battery system.

[0078] Various DNA tweezer constructs (FIGs. 2A - 3B) may be tested using in vitro fluorescence-based DNA strand displacement techniques and analyses followed by ex vivo validation in a FET transistor. Selected genomic sequence probes may be optimizable for successful hybridization events resulting in identifiable electrical charges, but in conditions that mimic central venous flow conditions.

[0079] Experimental methods with respect to various potential embodiments:

[0080] Materials: All DNA oligonucleotides candidates may be purchased from IDT (Coralville, IA) for all DNA sequences, and may be validated in vitro (e.g., using both fluorescence and electrical detection systems) for performance. Probes may be modified and optimized for flow conditions, including the toe-hold length and toe-hold position.

[0081] Fluorescence validation of Probes: The fluorescence quencher tagged normal strand (N), and Texas red-labeled weak strand (W) may be mixed in 1 : 1 ratio in PBS buffer. The N and W strand annealing process may be performed. The hybridization kinetic of perfect match strand and single mismatch strand with the probe (DS) may be performed by the exciting sample at 590 nm and emission recorded at 620 nm. A customized microfluidics device / mask may be created for testing probes. We may conjugate the Texas red tagged DNA (without quencher) to analyze the effect of flow on the detachment of probe from the GFET surface by analyzing the fluorescence of aliquot obtained from microfluidics outlet. We may mimic physiological conditions in the microfluidic device to mimic central venous blood flow.

[0082] Immobilization of DNA tweezers: To immobilize and conjugate candidate probes to the graphene surface, the graphene may be treated for one hour using pyrenebutanoic acid succinimidyl ester (PBASE) (5 mM) dissolved in dimethylformamide (DMF) and rinsed with DMF as well as DI water. The amino-modified DNA tweezers probes may be conjugated on the PBASE modified graphene surface. The unsaturated amino group on PBASE may be treated using ethanolamine solution and further rinsed by using lx PBS buffer. PBASE makes pi-pi bonds with graphene on the aromatic side, while the aliphatic region has activated carboxyl groups that make peptide bonds with the amine-functionalized probes. Multimode AFM equipped with a Nanoscope V controller (Bruker) may be used to acquire DNA topographic images on the graphene surface.

[0083] Strand Displacement on the Chip: The strand displacement reaction may be performed using perfect match single mismatch in the range of 100 pM-100 nM in 50 pL reaction volume. The strand incubated with probe and shift in Dirac voltage analyzed with respect to time and concentration of target.

[0084] Electrical Measurements: A semiconductor parameter analyzer equipped with a probe station may be used to measure the I-V curves and resistance. To apply gate voltage (Vg) through 12.5 mM MgC12/30 mM Tris buffer solution, silver wire may be used, and Vg may be swept from -0.5 to 1 V. Drain-source current (Ids) may be measured at an assigned Vds. The resistance measurements may be performed using a standard digital multi-meter (DMM) (Fluke 175 True RMS multimeter). The source voltage may be swept between 0 - 0.5 V. Current values may be converted into a voltage signal by introducing a pull-up resistor of 1 kQ resulting in the potential difference (V=IR) 1000 times the current measured by the device. Voltage values may be plotted against their respective current values with a trend line. The gradient of the trend line represents the resistance of the entire circuit with the resistance of the device obtained by subtracting the resistance of the other components, 1 kQ pull-up resistor and 10 kQ filtering resistor. To reset annealing of the tweezer strands (and liberate the opening strand ctDNA), a reverse current (nA) may be sent through the metal/graphene junctions (which will act as resistors) on the micro-circuit to heat the graphene surfaces of the FETs to achieve melting temperatures.

[0085] Specific third aspect: Testing and validation of a prototype implantable ctDNA sensor in vitro and in vivo. With a powerized FET microcircuit, and after optimizing candidate DNA tweezers in vitro, we may attach the probes to the graphene FET islands on the microcircuit designed for the VAP (see experimental methods for aspect 1 and aspect 2) and this prototype may be evaluated for performance in an in vitro system that mimics vascular flow conditions. There are several devices such as vascular bypass devices and out- of-service dialysis machines that can be modified for this purpose. Synthesized ctDNA oligonucleotides may be added to the system to evaluate electrical signal detection of the prototype devices. The prototype may trigger electrical signals in response to ctDNA <0.01% of total cfDNA. [0086] Experimental methods: Miniaturization of the single sensor and multiplexed DNA array on an arrayed FET microcircuit chip: Optimal methodology for multiplexing DNA probes to the FET microcircuit chip may be developed based on above principles. Fabrication of arrayed graphene FETs may begin by transferring monolayer of graphene sheet onto a Si02/Si substrate. The graphene sheet may then be patterned into an array of graphene sites by micro-lithography and 02 plasma etching. Source and drain contacts (using appropriate metals) may be made on both ends of each graphene site. The metal contacts may be passivated by depositing a Si02 layer to prevent the leakage current from the metal contacts to the electrolyte.

[0087] In some embodiments of the system of the present technology, the set of DNA probes are configured to bind to ctDNA comprising at least one mutation in one or more cancer- related genes present in a cancer patient. In certain embodiments, the set of DNA probes are configured to bind to ctDNA comprising at least one mutation and/or at least one rearrangement in one or more cancer-related genes. Examples of mutations in cancer-related genes include, for example: AKT1, ALK, APC, AR, ARAF, ARID 1 A, ARID2, ATM, B2M, BCL2, BCOR, BRAF, BRCA1, BRCA2, CARD11, CBFB, CCNDl, CDH1, CDK4, CDKN2A, CIC, CREBBP, CTCF, CTNNBl, DICERl, DIS3, DNMT3A, EGFR, EIF1AX, EP300, ERBB2, ERBB3, ERCC2, ESR1, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FGFR4, FLT3, FOXA1, FOXL2, FOXOl, FUBP1, GAT A3, GNA11, GNAQ, GNAS, H3F3A, HIST1H3B, HRAS, IDH1, IDH2, IKZF1, INPPLl, JAK1, KDM6A, KEAPl, KIT,

KNSTRN, KRAS, MAP2K1, MAPKl, MAX, MED 12, MET, MLHl, MSH2, MSH3,

MSH6, MTOR, MYC, MYCN, MYD88, MYOD1, NFl, NFE2L2, NOTCH1, NRAS, NTRKl, NTRK2, NTRK3, NUP93, PAK7, PDGFRA, PIK3CA, PIK3CB, PIK3R1, PIK3R2, PMS2, POLE, PPP2R1A, PPP6C, PRKCI, PTCH1, PTEN, PTPN11, RAC1, RAFl, RBI, RET, RHOA, RIT1, ROS1, RRAS2, RXRA, SETD2, SF3B1, SMAD3, SMAD4,

SMARCA4, SMARCBl, SOS1, SPOP, STAT3, STK11, STK19, TCF7L2, TGFBR1, TGFBR2, TP53, TP63, TSC1, TSC2, U2AF1, VHL, XPOl, and TERT. Examples of rearrangements in cancer-related genes include, for example: ALK, BRAF, EGFR, ETV6, FGFR2, FGFR3, MET, NTRKl, RET, and ROSE

[0088] Various potential embodiments include the following examples. It is noted that elements or features of any embodiments disclosed in this document can be combined in any suitable manner with any other elements or features of any other embodiments without limitation.

[0089] Embodiment A: A vascular access port system comprising an implantable indwelling device for in-situ detection of circulating cell-free tumor DNA (ctDNA), the indwelling device comprising: a reservoir and a catheter extending from the reservoir, the catheter being configured to interface with a circulatory system of a subject to access a bodily fluid in the circulatory system of the subject; and a DNA detector comprising: a set of one or more DNA probes positioned at the catheter and/or reservoir, the set of DNA probes comprising at least a normal DNA strand complementary to a ctDNA strand; and a set of one or more sensors configured to detect signals corresponding to DNA hybridizations at one or more DNA probes in the set of DNA probes.

[0090] Embodiment B: The system of Embodiment A, wherein the set of DNA probes comprises a first DNA probe with a first normal DNA strand complementary to a first ctDNA strand, and a second DNA probe with a second normal DNA strand complementary to the first ctDNA strand or to a second ctDNA strand.

[0091] Embodiment C: The system of either Embodiment A or B, wherein the set of sensors comprises at least a first sensor situated at the first DNA probe and configured to detect signals corresponding to hybridization of the first normal DNA strand to the first ctDNA strand, and a second sensor situated at the second DNA probe and configured to detect signals corresponding to hybridization of the second normal DNA strand to the first ctDNA strand or to the second ctDNA strand.

[0092] Embodiment D: The system of any of Embodiments A - C, wherein the normal strand comprises a receptive region complementary to the ctDNA strand, and wherein the receptive region comprises DNA bases selected from a first group of DNA bases consisting of adenine (A), guanine (G), cytosine (C), and thymine (T).

[0093] Embodiment E: The system of any of Embodiments A - D, wherein the set of DNA probes further comprises a weak strand complementary to the receptive region of the normal strand, and wherein the weak strand comprises DNA bases selected from a second group of DNA bases consisting of adenine (A), guanine (G), inosine (I), cytosine (C) and thymine (T). [0094] Embodiment F: The system of Embodiment E, wherein the weak strand is configured to decouple from the normal strand in the presence of the ctDNA strand.

[0095] Embodiment G: The system of any of Embodiments A - F, wherein the indwelling device comprises a graphene surface to which the normal strand is secured.

[0096] Embodiment H: The system of any of Embodiments A - G, wherein at least one sensor in the set of sensors comprises a field effect transistor (FET) configured to detect electrical signals from hybridizations at one or more DNA probes in the set of DNA probes.

[0097] Embodiment F The system of Embodiment H, wherein at least one FET in the set of sensors is a graphene FET (GFET).

[0098] Embodiment J: The system of Embodiment H, wherein the indwelling device further comprises a microprocessor coupled, via electrodes, to source and drain terminals of the FET.

[0099] Embodiment K: The system of any of Embodiments A - J, wherein the DNA detector comprises a first graphene cuff and a second graphene cuff, each cuff extending from a first end to a second end, wherein the first and second graphene cuffs encircle an external surface of the catheter without the first and second ends of the cuffs making contact with each other circumferentially, and wherein the first and second graphene cuffs comprise two or more DNA probes in the set of DNA probes.

[0100] Embodiment L: The system of any of Embodiments A - K, wherein the indwelling device further comprises a wireless transmitter configured to wirelessly transmit signals detected via the set of sensors to a remote computing device.

[0101] Embodiment M: The system of any of Embodiments A - L, wherein the indwelling device further comprises a thermal element coupled to a controller configured to use the thermal element to heat one or more DNA probes in the set of DNA probes to a melting temperature at which the normal strand liberates the ctDNA strand.

[0102] Embodiment N: The system of any of Embodiments A - M, wherein the indwelling device further comprising a power supply comprising a battery and a charging module coupled thereto, wherein the charging module is an inductive charging module situated at a diaphragm of the reservoir. [0103] Embodiment O: The system of any of Embodiments A -N, wherein the DNA detector is configured to detect one or more variants identified in a tumor of the subject.

[0104] Embodiment P: A method of detecting circulating cell-free tumor DNA (ctDNA) in situ in a subject using a vascular access port system, the method comprising implanting an indwelling device in the subject, the indwelling device comprising: a reservoir and a catheter extending from the reservoir, the catheter being configured to interface, when implanted, with a circulatory system of the subject to access the subject’s blood; a DNA detector comprising: a set of DNA probes situated on the catheter and/or reservoir, the set of DNA probes comprising at least a first DNA probe with a first normal DNA strand complementary to a first ctDNA strand, and a second DNA probe with a second normal DNA strand complementary to the first ctDNA strand or to a second ctDNA strand; a set of sensors configured to detect electrical signals corresponding to DNA hybridizations at the DNA probes, the set of sensors comprising at least a first sensor situated at the first DNA probe and configured to detect signals corresponding to hybridization of the first normal DNA strand to the first ctDNA strand, and a second sensor situated at the second DNA probe and configured to detect signals corresponding to hybridization of the second normal DNA strand to the first ctDNA strand or to the second ctDNA strand; and a wireless transmitter configured to transmit detected signals to a remote computing device.

[0105] Embodiment Q: The method of Embodiment P, further comprising wirelessly receiving, at the remote computing device, signals from the indwelling device, and determining, based on the signals, that one of the first ctDNA strand or the second ctDNA strand is present in the subject’s blood.

[0106] Embodiment R: The method of either Embodiment P or Q, wherein the indwelling device further comprises a power supply comprising a battery and an inductive charging module coupled thereto, the inductive charging module situated at a diaphragm of the reservoir, and wherein the method further comprises inductively charging the battery of the indwelling device by positioning an inductive charger near the implanted reservoir of the indwelling device.

[0107] Embodiment S: The method of any of Embodiments P - R, wherein: the first DNA probe further comprises a first weak strand complementary to a first receptive region of the first normal strand, the first weak strand configured to decouple from the first normal strand in the presence of the first ctDNA strand; the second DNA probe further comprises a second weak strand complementary to a second receptive region of the second normal strand, wherein the second weak strand is configured to decouple from the second normal strand in the presence of the second ctDNA strand; the DNA detector further comprises a thermal element; and the method further comprises heating, via the thermal element, the first and/or the second DNA probes to a melting temperature at which the first normal strand and/or the second normal strand liberates the first ctDNA strand and/or the second ctDNA strand, respectively.

[0108] Embodiment T: The method of any of Embodiments P - S, wherein: The method of claim 17, further comprising sequencing a tumor of the subject to identify one or more variants in the tumor not present in germline control cells, wherein the DNA detector is configured to detect the one or more variants.

[0109] Additional enabling details can be found in the following references (which are referenced above in parentheses):

[0110] 1 Chin, R. I. et al. Detection of Solid Tumor Molecular Residual Disease

(MRD) Using Circulating Tumor DNA (ctDNA). Mol Diagn Ther 23, 311-331, doi : 10.1007/s40291 -019-00390-5 (2019).

[0111] 2 Jahr, S. et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res 61, 1659-1665 (2001).

[0112] 3 Jiang, P. & Lo, Y. M. D. The Long and Short of Circulating Cell-Free DNA and the Ins and Outs of Molecular Diagnostics. Trends Genet 32, 360-371, doi: 10.1016/j .tig.2016.03.009 (2016).

[0113] 4 Underhill, H. R. et al. Fragment Length of Circulating Tumor DNA. PLoS

Genet 12, el006162, doi: 10.1371/journal. pgen.1006162 (2016).

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Label-Free Genotyping. Adv Mater, el802440, doi:10.1002/adma.201802440 (2018). [0136] As utilized herein, the terms “approximately,” “about,” “substantially”, and similar terms are intended to have a broad meaning in harmony with the common and accepted usage by those of ordinary skill in the art to which the subject matter of this disclosure pertains. It should be understood by those of skill in the art who review this disclosure that these terms are intended to allow a description of certain features described and claimed without restricting the scope of these features to the precise numerical ranges provided. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and claimed are considered to be within the scope of the disclosure as recited in the appended claims.

[0137] It should be noted that the terms “exemplary,” “example,” “potential,” and variations thereof, as used herein to describe various embodiments, are intended to indicate that such embodiments are possible examples, representations, or illustrations of possible embodiments (and such terms are not intended to connote that such embodiments are necessarily extraordinary or superlative examples).

[0138] The term “coupled” and variations thereof, as used herein, means the joining of two members directly or indirectly to one another. Such joining may be stationary (e.g., permanent or fixed) or moveable (e.g., removable or releasable). Such joining may be achieved with the two members coupled directly to each other, with the two members coupled to each other using a separate intervening member and any additional intermediate members coupled with one another, or with the two members coupled to each other using an intervening member that is integrally formed as a single unitary body with one of the two members. If “coupled” or variations thereof are modified by an additional term (e.g., directly coupled), the generic definition of “coupled” provided above is modified by the plain language meaning of the additional term (e.g., “directly coupled” means the joining of two members without any separate intervening member), resulting in a narrower definition than the generic definition of “coupled” provided above. Such coupling may be mechanical, electrical, or fluidic.

[0139] The term “or,” as used herein, is used in its inclusive sense (and not in its exclusive sense) so that when used to connect a list of elements, the term “or” means one, some, or all of the elements in the list. Conjunctive language such as the phrase “at least one of X, Y, and Z,” unless specifically stated otherwise, is understood to convey that an element may be either X, Y, Z; X and Y; X and Z; Y and Z; or X, Y, and Z (i.e., any combination of X, Y, and Z). Thus, such conjunctive language is not generally intended to imply that certain embodiments require at least one of X, at least one of Y, and at least one of Z to each be present, unless otherwise indicated.

[0140] References herein to the positions of elements (e.g., “top,” “bottom,” “above,” “below”) are merely used to describe the orientation of various elements in the Figures. It should be noted that the orientation of various elements may differ according to other exemplary embodiments, and that such variations are intended to be encompassed by the present disclosure.

[0141] The embodiments described herein have been described with reference to drawings. The drawings illustrate certain details of specific embodiments that implement the systems, methods and programs described herein. However, describing the embodiments with drawings should not be construed as imposing on the disclosure any limitations that may be present in the drawings.

[0142] While various inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other mechanisms and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that, unless otherwise noted, any parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein.

It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. [0143] Also, the technology described herein may be embodied as a method, of which at least one example has been provided. The acts performed as part of the method may be ordered in any suitable way unless otherwise specifically noted. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

[0144] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of’ will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0145] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one,

B (and optionally including other elements); etc. [0146] The foregoing description of embodiments has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosure to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from this disclosure. The embodiments were chosen and described in order to explain the principals of the disclosure and its practical application to enable one skilled in the art to utilize the various embodiments and with various modifications as are suited to the particular use contemplated. Other substitutions, modifications, changes and omissions may be made in the design, operating conditions and arrangement of the embodiments without departing from the scope of the present disclosure as expressed in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A vascular access port system comprising an implantable indwelling device for in-situ detection of circulating cell-free tumor DNA (ctDNA), the indwelling device comprising: a reservoir and a catheter extending from the reservoir, the catheter being configured to interface with a circulatory system of a subject to access a bodily fluid in the circulatory system of the subject; and a DNA detector comprising: a set of one or more DNA probes positioned at the catheter and/or reservoir, the set of DNA probes comprising at least a normal DNA strand complementary to a ctDNA strand; and a set of one or more sensors configured to detect signals corresponding to DNA hybridizations at one or more DNA probes in the set of DNA probes.

2. The system of claim 1, wherein the set of DNA probes comprises a first DNA probe with a first normal DNA strand complementary to a first ctDNA strand, and a second DNA probe with a second normal DNA strand complementary to the first ctDNA strand or to a second ctDNA strand.

3. The system of claim 2, wherein the set of sensors comprises at least a first sensor situated at the first DNA probe and configured to detect signals corresponding to hybridization of the first normal DNA strand to the first ctDNA strand, and a second sensor situated at the second DNA probe and configured to detect signals corresponding to hybridization of the second normal DNA strand to the first ctDNA strand or to the second ctDNA strand.

4. The system of claim 1, wherein the normal strand comprises a receptive region complementary to the ctDNA strand, and wherein the receptive region comprises DNA bases selected from a first group of DNA bases consisting of adenine (A), guanine (G), cytosine (C), and thymine (T).

5. The system of claim 4, wherein the set of DNA probes further comprises a weak strand complementary to the receptive region of the normal strand, and wherein the weak strand comprises DNA bases selected from a second group of DNA bases consisting of adenine (A), guanine (G), inosine (I), cytosine (C) and thymine (T).

6. The system of claim 5, wherein the weak strand is configured to decouple from the normal strand in the presence of the ctDNA strand.

7. The system of claim 1, wherein the indwelling device comprises a graphene surface to which the normal strand is secured.

8. The system of claim 1, wherein at least one sensor in the set of sensors comprises a field effect transistor (FET) configured to detect electrical signals from hybridizations at one or more DNA probes in the set of DNA probes.

9. The system of claim 8, wherein at least one FET in the set of sensors is a graphene FET (GFET).

10. The system of claim 8, wherein the indwelling device further comprises a microprocessor coupled, via electrodes, to source and drain terminals of the FET.

11. The system of claim 1, wherein the DNA detector comprises a first graphene cuff and a second graphene cuff, each cuff extending from a first end to a second end, wherein the first and second graphene cuffs encircle an external surface of the catheter without the first and second ends of the cuffs making contact with each other circumferentially, and wherein the first and second graphene cuffs comprise two or more DNA probes in the set of DNA probes.

12. The system of claim 1, wherein the indwelling device further comprises a wireless transmitter configured to wirelessly transmit signals detected via the set of sensors to a remote computing device.

13. The system of claim 1, wherein the indwelling device further comprises a thermal element coupled to a controller configured to use the thermal element to heat one or more DNA probes in the set of DNA probes to a melting temperature at which the normal strand liberates the ctDNA strand.

14. The system of claim 1, wherein the indwelling device further comprising a power supply comprising a battery and a charging module coupled thereto, wherein the charging module is an inductive charging module situated at a diaphragm of the reservoir.

15. The system of claim 1, wherein the DNA detector is configured to detect one or more variants identified in a tumor of the subject.

16. A method of detecting circulating cell-free tumor DNA (ctDNA) in situ in a subject using a vascular access port system, the method comprising implanting an indwelling device in the subject, the indwelling device comprising: a reservoir and a catheter extending from the reservoir, the catheter being configured to interface, when implanted, with a circulatory system of the subject to access the subject’s blood; a DNA detector comprising: a set of DNA probes situated on the catheter and/or reservoir, the set of DNA probes comprising at least a first DNA probe with a first normal DNA strand complementary to a first ctDNA strand, and a second DNA probe with a second normal DNA strand complementary to the first ctDNA strand or to a second ctDNA strand; a set of sensors configured to detect electrical signals corresponding to DNA hybridizations at the DNA probes, the set of sensors comprising at least a first sensor situated at the first DNA probe and configured to detect signals corresponding to hybridization of the first normal DNA strand to the first ctDNA strand, and a second sensor situated at the second DNA probe and configured to detect signals corresponding to hybridization of the second normal DNA strand to the first ctDNA strand or to the second ctDNA strand; and a wireless transmitter configured to transmit detected signals to a remote computing device.

17. The method of claim 15, further comprising wirelessly receiving, at the remote computing device, signals from the indwelling device, and determining, based on the signals, that one of the first ctDNA strand or the second ctDNA strand is present in the subject’s blood.

18. The method of claim 15, wherein the indwelling device further comprises a power supply comprising a battery and an inductive charging module coupled thereto, the inductive charging module situated at a diaphragm of the reservoir, and wherein the method further comprises inductively charging the battery of the indwelling device by positioning an inductive charger near the implanted reservoir of the indwelling device.

19. The method of claim 15, wherein: the first DNA probe further comprises a first weak strand complementary to a first receptive region of the first normal strand, the first weak strand configured to decouple from the first normal strand in the presence of the first ctDNA strand; the second DNA probe further comprises a second weak strand complementary to a second receptive region of the second normal strand, wherein the second weak strand is configured to decouple from the second normal strand in the presence of the second ctDNA strand; the DNA detector further comprises a thermal element; and the method further comprises heating, via the thermal element, the first and/or the second DNA probes to a melting temperature at which the first normal strand and/or the second normal strand liberates the first ctDNA strand and/or the second ctDNA strand, respectively.

20. The method of claim 15, further comprising sequencing a tumor of the subject to identify one or more variants in the tumor not present in germline control cells, wherein the DNA detector is configured to detect the one or more variants.