CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/305,884, filed on February 2, 2022, which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Accurate and reliable testing is crucial in any strategy for disease control, treatment, and prevention. Advances in diagnostics can potentially set the pace of modern healthcare settings and global health. The development of rapid diagnostics has been identified as a strategic priority by the White House, the US Department of Health and Human Services (HHS), the National Institutes of Health (NIH), the US Centers for Disease Control and Prevention (CDC), and the US Department of Defense (DoD). The early diagnosis and timely initiation of treatment remain the key strategy for disease management and prevention. One category of diagnostic technologies - rapid point-of-care (POC) tests - offers several possible advantages over other diagnostic approaches in this perspective. POC diagnostics have the potential to expedite clinical decision-making, reduce patient loss to follow-up while waiting for test results, and facilitate the deliver}' of care outside traditional healthcare settings. POC diagnostics are also appealing for use in areas with physician shortages and lower-cost environments because they require less complicated infrastructure and training.

[0003] POC diagnostics have proven clinically useful for disease testing - easy to transport, operate, and maintain. Furthermore, they are sensitive, reliable, and less-skilled staff are equally able to perform these tests compared to trained laboratory technicians - eliminating most of the limitations associated with the standard techniques currently used for disease testing, including ELISA and PCR techniques. Kozel, T.R. and A.R. Burnham-Marusich, J Clin Microbiol., 55(8): p. 2313-2320 (2017). Nucleic acid testing (NAT) is the most accurate and preferred approach for the molecular detection of disease biomarkers and health conditions (e.g., infectious diseases, cancer, autoimmune, respiratory, cardiovascular, and neurodegenerative diseases, etc.), and is highly recommended by WHO as an essential tool for healthcare and disease management, and prevention. Tang et al., Clin Chem., 43(11): p. 2021-

SUBSTITUTE SHEET ( RULE 26) 2038 (1997). POC diagnostics that allow for nucleic testing will enable individuals to effectively self-manage their health, day-to-day risk, and treatment with high accuracy and sensitivity. PCR-based methods remain the “gold standard” for NAT, disease diagnosis, and treatment monitoring. However, due to the need for expensive and bulky thermal cycling equipment and highly trained personnel, it cannot readily be implemented at POC and selfadministered settings.

SUMMARY OF THE INVENTION

[0004] Here, and building on our experience with the development of neural network-enabled smartphones using nanoparticles for virus detection (Draz et al., Sci Adv., 6(51):eabd5354 (2020), we developed a novel platform technology that is inexpensive, portable, and allows for accurate, rapid, and highly sensitive nucleic acid testing in small- volume samples.

[0005] Our technology leverages cutting-edge advances in DNA engineering, microfluidics, nanotechnology, image processing, and computing to allow a rapid and simple optical detection of nucleic acid using a cellphone. The centerpiece of this technology is a novel target-triggered DNA locking approach that utilizes uniquely designed lock-in DNA amplifiers to direct the amplification of the target nucleic acid into catalytically active 3D DNA structures on the surface of a chip. The lock-in DNA amplifiers are head-tailed structures in which the head is prepared with readily amplifiable reference gene sequence and tails that include target- specific sequences. The locking of the target nucleic acid between the tails of the amplifiers triggers the isothermal amplification of the head reference gene sequence using biotin-modified looping primers to form highly looped amplicons. The formed DNA amplicons are biotinylated to selfassemble with streptavidin-conjugated PtNPs into PtNP-DNA structures that are large enough to induce the formation of visible bubbles in the presence of peroxide solution. The formed bubbles can be optically detected using a cellphone enabled with an Al-based imaging algorithm. Bubbles as a readout are unique — the localized nature of gas signals in the form of bubbles makes the visual detection of the target simpler and more sensitive when compared with other sensing approaches that utilize color (e.g., ELISA) and fluorescence (e.g., PCR) labels. In which the signals tend to diffuse in a relatively large volume of sample that leads to a lower detection limit and sensing efficiency. Furthermore, bubbles enable the easy and efficient integration of microchips with cellphones to develop a hardware-free system for nucleic acid (NA) detection, which is a major advancement in the development of mobile health

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SUBSTITUTE SHEET ( RULE 26) and POC technologies. The developed Al-based image analysis system is a powerful universal modality that can be adapted to any cellphone model without the need for extensive modifications. Such advancement is a major step towards true hardware-free cellphone diagnostics by uniquely integrating microfluidics, DNA engineering, nanotechnology, and Al.

BRIEF DESCRIPTION OF THE FIGURES

[0006] The present invention may be more readily understood by reference to the following drawings wherein:

[0007] Figure 1 provides a schematic representation of Schematic representation of LAMP reaction and its principle. The design of primer for LAMP reaction and is characterized by the use of four different primers specially designed to recognize six distinct regions of the target DNA. Forward Inner Primer (FIP) consists of a F2 region at the 3'-end and an Flc region at the 5 '-end; Forward Outer Primer (F3 Primer) consists of a F3 region which is complementary to the F3c region of the template sequence; Backward Inner Primer (BIP) consists of a B2 region at the 3'-end and a Bic region at the 5'-end. Backward Outer Primer (B3 Primer) consists of a B3 region which is complementary to the B3c region of the template sequence. LAMP reaction: The amplification starts when F2 region of FIP hybridizes to F2c region of the target DNA and initiates complementary strand synthesis, follow by F3 primer that hybridizes to the F3c region of the target DNA and extends, displacing the FIP linked complementary strand. This displaced strand forms a loop at the 5'-end. This single-stranded DNA with a loop at the 5’-end then serves as a template for BIP. B2 hybridizes to B2c region of the template DNA. DNA synthesis is initiated leading to the formation of a complementary strand and opening of the 5 '-end loop. Subsequently, B3 hybridizes to B3c region of the target DNA and extends, displacing the BIP linked complementary strand. This results in the formation of a dumbbell-shaped DNA. The nucleotides are added to the 3'-end of Fl by Bst DNA polymerase, which extends and opens up the loop at the 5 '-end. The dumbbell -shaped DNA now gets converted to a stem-loop structure (refer a and b). This structure serves as an initiator for LAMP cycling, which is the second stage of the LAMP reaction. Loop primers can be added as well for exponential amplification of LAMP. The final products obtained are a mixture of stem-loop DNA with various stem lengths and various cauliflower-like structures with multiple loops.

[0008] Figure 2 provides a block diagram of a system for detection of target polynucleotides and a neural network-enabled mobile device.

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SUBSTITUTE SHEET ( RULE 26) [0009] Figure 3 provides a schematic representation of nucleic acid amplification, capturing, and labeling on microchip. Nucleic acid is first amplified using DNA amplifiers specifically designed with reference gene sequence that results into the formation of biotinylated large DNA amplicons that are loaded into streptavidin (SA) -functionalized microchips. The captured LAMP amplicons are then labeled with Pt- nanoprobes.

[0010] Figures 4a-4c provide schematics of microchip fabrication and surface functionalization, (a) Schematic of microchip fabrication and functionalization. Microchips are fabricated using polymethyl methacrylate (PMMA), double-sided adhesive (DSA), and a glass slide. The glass surface of microchip is first silanized with silane-polyethylene glycol-thiol (Si- PEG-SH) that then coupled to maleimide-modified streptavidin (SA) for capturing of biotinylated DNA (b) FT-IR spectra of surface functionalized chips, (c) SDS-PAGE analysis of SA and mAb (as a model protein) released from the surface of microchip.

[0011] Figures 5a-5c provide graphs and images confirming the results of Pt-nanoprobe preparation, (a) Characterization of PtNPs: TEM image and particle size distribution histogram, (b) UV-vis absorption spectra of PtNPs (citrate capped platinum nanoparticles, black), and Pt- nanoprobe with SA (platinum nanoparticles conjugated to SA). The insert is a schematic of Pt- nanoprobes preparation. Thiol- streptavidin (SA) is directly conjugated to the surface of platinum nanoparticles (PtNPs). (c) Agarose gel electrophoresis of PtNPs (unmodified platinum nanoparticles) and Pt-nanoprobes with SA (SA-conjugated platinum nanoparticles).

[0012] Figures 6a-6e provide images of HIV-1 RNA amplification, capturing, and bubble formation on a chip, (a) Electrophoresis patterns of DNA amplicons, (b) Concentration of biotinylated DNA amplicons captured on the surface of streptavidin (SA)-modified and nonmodified (no SA) chips, measured using direct absorbance at 280 nm. (c) Fluorescence imaging of DNA amplicons (0-100% of amplification product diluted in PBS buffer) captured using SA-modified chip, confirming the ability of the chip to specifically interact with the biotinylated DNA amplicons, (d) Bright-field images of bubbles after capturing and labeling of biotinylated DNA with Pt-nanoprobes (with SA) at 15-180 s. (e) The increase in bubble number with the increase of incubation time in peroxide solution (10% H2O2 and 20% glycerol) was added on the chip with the 3D PtNP-DNA structures.

[0013] Figures 7A and 7B provide A) images showing the performance of the developed microchip in the detection of HIV-1 RNA in PBS samples. Agarose gel electrophoresis of

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SUBSTITUTE SHEET ( RULE 26) LAMP amplification products of different RNA concentrations spiked in PBS buffer. Representative images of chips associated with HIV-spiked PBS samples, and B) images showing bubble formation in the presence of non-biotinylated DNA amplicons, biotinylated DNA amplicons, and biotin-spiked blood samples.

[0014] To illustrate the invention, several embodiments of the invention will now be described in more detail. Reference will be made to the drawings, which are summarized above. Skilled artisans will recognize the embodiments provided herein have many useful alternatives that fall within the scope of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0015] This disclosure provides a device for detecting a target polynucleotide in a sample. The device includes a microchip comprising at least one channel configured to receive a sample comprising a target polynucleotide, one or more LAMP probes bound to a surface of the channel that specifically bind to the one or more target polynucleotides, a source of detectable nanoparticles that bind to the LAMP probes, and a detector configured to detect a signal created by the detectable nanoparticles. Systems including the device together with a mobile device, and methods of using the device to detect a target polynucleotide in a sample are also described. Systems including the device together with a mobile device, and methods of using the device to detect a target polynucleotide in a sample are also provided.

Definitions

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present specification, including definitions, will control.

[0017] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting of the invention as a whole. Unless otherwise specified, "a," "an," “"the," and "at least one" are used interchangeably. Furthermore, as used in the description of the invention and the appended claims, the singular forms “a”, “an”, and “the” are inclusive of their plural forms, unless contraindicated by the context surrounding such.

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SUBSTITUTE SHEET ( RULE 26) [0018] Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value, except that the value will never deviate by more than 5% from the value cited.

[0019] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0020] "Nucleic acid" or "oligonucleotide" or "polynucleotide", as used herein, may mean at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. The term “nucleotide sequence,” as used herein, refers to an oligonucleotide, nucleotide, or polynucleotide of single-stranded or double stranded DNA or RNA, or fragments thereof.

[0021] A "subject", as used herein, refers to an animal such as a vertebrate or invertebrate animal. In some embodiments, the subject is a mammal, including, but not limited to, primates, including simians and humans, equines (e.g., horses), canines (e.g., dogs), felines, various domesticated livestock (e.g., ungulates, such as swine, pigs, goats, sheep, and the like), as well as domesticated pets and animals maintained in zoos. In some embodiments, the subject is a human subject.

Target Polynucleotide Detection Devices

[0022] One aspect of the invention provides a device for detecting a target polynucleotide in a sample. The device includes a microchip comprising at least one channel configured to receive a sample comprising one or more target polynucleotides, one or more LAMP probes bound to a surface of the channel that specifically bind to the one or more target polynucleotides, a

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SUBSTITUTE SHEET ( RULE 26) source of detectable nanoparticles that bind to the LAMP probes, and a detector configured to detect a signal created by the detectable nanoparticles.

[0023] The microchip is configured to receive a sample (e.g., blood, plasma/serum, serum) to determine if the sample includes the one or more target polynucleotides. In some embodiments, the microchip is a single channel microchip and the sample is loaded into the microchip (e.g., using a pipette). In some embodiments, the single channel microchip may be fabricated from glass slides and layers of poly(methyl methacrylate) (PMMA), and double-sided adhesive film (DS A). The surface of the microchip is modified with one ore more LAMP probes to capture any target polynucleotides in the sample. In addition, the sample can be processed on the microchip using, for example, nanoparticles and a hydrogen peroxide solution, to allow for the formation of bubbles on the surface of the microchip in the presence of a target polynucleotide in the sample.

[0024] LAMP probes may be conjugated to the surface of the chips using a surface chemistry protocol specifically designed to allow efficient directional conjugation of the nucleotides with terminal thiol and silane group. For example, a direct conjugation of thiol-activated streptavidin (SA) to the surface of citrate PtNPs, by the well-known thiol-metal bond can be used to bind the LAMP probes to the surface of the channel. The microchip of the device includes one or more LAMP probes bound to a surface of the channel that specifically bind to the one more target polynucleotides.

[0025] A LAMP probe "specifically binds" when the probe preferentially binds a target structure, or subunit thereof, but binds to a substantially lesser degree or does not bind to a biological molecule that is not a target structure. In some embodiments, the probe specifically binds to the target polynucleotide with a specific affinity of between 10’ ⁸ M and 10 ¹¹ M. In some embodiments, the probe binds to the target analyte with a specific affinity of greater than IO’ ⁷ M, IO’ ⁸ M, IO’ ⁹ M, IO’ ¹⁰ M, or 10 ¹¹ M, between IO’ ⁸ M - 10 ¹¹ M, IO’ ⁹ M - IO’ ¹⁰ M, and IO ¹⁰ M - 10 ¹¹ M. Specific binding is measured using a competitive binding assay as set forth in Ausubel F.M., (1994). Current Protocols in Molecular Biology. Chichester: John Wiley and Sons ("Ausubel"), which is incorporated herein by reference.

[0026] The device one or more loop-mediated isothermal amplification (LAMP) probes bound to a surface of the channel that specifically bind to the target polynucleotide. LAMP probes were first described in 2000. Notomi et al., Nucleic Acids Res., 15 ;28(12) :E63 (2000). For a

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SUBSTITUTE SHEET ( RULE 26) more detailed review of LAMP, see Wong et al., J Appl Microbiol., 124(3): 626-643 (2018), the disclosure of which is incorporated herein by reference. The LAMP technique is one in which a set of four (or six) different primers within the LAMP probe bind to six (or eight) different regions on the target gene, making it highly specific. The probe consists of two outer primers, two inner primers, and loop primers. A schematic representation of the LAMP reaction and the structure of the LAMP probe are shown in Figure 1. A variety of additional types of LAMP have been developed since its initial discovery by Notomi et al. These include conventional LAMP, reverse-transcription LAMP, and multiplex LAMP.

[0027] In some embodiments, the nanoparticles are metal nanoparticles. The metal nanoparticle can be made of any metal. Metal groups suitable for the metal nanoparticles included in the nanocomposites of the present invention include transition metals (e.g., gold, silver and platinum), post-transition metals (e.g., aluminum, indium and lead), lanthanides (e.g., cerium, gadolinium and terbium), and actinides (e.g., uranium, thorium and plutonium). In some embodiments, the metal nanoparticle is selected from the group consisting of gold, silver, copper, platinum, iron, and palladium nanoparticles. A preferred metal nanoparticle is a platinum nanoparticle (PtNP).

[0028] The metal nanoparticle size can range from 0.1 nm to 150 nm. Other suitable ranges include 0.5 nm to 100 nm, 0.5 nm to 50 nm, and 1.0 nm to 50 nm. The metal nanoparticle can be in form of nanoclusters (0.1 - 10 nm), nanoparticles (5 nm - 150 nm), nanorods, or any other crystalline shape (e.g., hexagonal, octagonal bipyramid and cubic). Metal nanoclusters consist of a small number of metal atoms, typically in the tens. A preferred size range of the metal nanoparticles, particularly for antimicrobial application, is 0.5 nm - 5 nm.

[0029] In some embodiments, the metal nanoparticles comprise platinum capable of catalyzing oxygen gas formation from hydrogen peroxide. The catalytic activity of PtNPs for gas formation is directly related to the size of the PtNPs, and larger nanoparticles are expected to have higher activity. Mostafa et al., J. Am. Chem. Soc. 132, 15714-15719 (2010). However, relatively small size PtNPs (4.5 nm in diameter) can be used to avoid surface particle load limitation during virus labeling and to minimize the effect of hydrodynamic shear forces on the formed Pt-virus complexes during washing and labeling steps that may release the captured viruses on-chip. Tan et al., Microfluid Nanofluidics 14, 77-87 (2013). In addition, high catalytic activity of PtNPs in high concentration of H2O2 leads to rapid merging of the

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SUBSTITUTE SHEET ( RULE 26) generated bubbles and forming irregular bubble shapes, which can make accurate signal detection difficult. To avoid rapid bubble merging and to control the stability of the visual patterns on-chip after virus capture and signal amplification, glycerol (20% of the solution) can be used to increase the density of the catalyzer solution to -0.73 g/cm ³ . The formed bubbles reached to macroscopic sizes (up to 90 pm) after 300 s of incubation at 10% H2O2 solution without visible merging.

[0030] A catalyzer solution can be loaded onto the microchip to cause the formation of gas bubbles (i.e., bubble signal) if target polynucleotides are present on the microchip. In some embodiments, the catalyzer solution includes hydrogen peroxide (H2O2). In the presence of captured Pt-virus immunocomplexes, bubbles can be formed due to the catalytic activity of PtNPs in contact with H2O2. High catalytic activity of PtNPs in high concentrations of H2O2, however, can lead to rapid merging of the generated bubbles on the surface of the microchip and form irregular bubble shapes which can make accurate signal detection difficult. In some embodiments, to help avoid rapid bubble merging and to help control the stability of the visual patterns on-chip after virus capture and signal amplification, glycerol can be included in the catalyzer solution to increase the density of the catalyzer solution. In some embodiments, the catalyzer solution includes 5% H2O2 and 20% glycerol. The microchip, sample, nanoparticles and catalyzer solution can be incubated for a third predetermined period of time to allow for bubble formation on the surface of the microchip if there are bound target polynucleotides on the surface of the microchip. In some embodiments, the third predetermined time period is ten minutes.

[0031 ] The present invention provides a device for detecting a target polynucleotide. The target polynucleotide can be a sequence within a larger polynucleotide that is distinctive and can be used to identify the source of the polynucleotide. The length of the target polynucleotide of the present invention is not particularly limited, but can be about 10 to about 200 nucleotides, or can be, for example, about 100 nucleotides or less, about 50 nucleotides or less, about 40 nucleotides or less, or about 35 nucleotides or less.

[0032] As can be seen in Figure 1, a LAMP probe includes multiple primer nucleotides that can bind to different regions of a target gene, providing for increased affinity and/or specificity. Accordingly, binding one or more target polynucleotides can refer to the binding of a LAMP probe to multiple polynucleotides in a target gene. However, if a plurality of LAMP probes

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SUBSTITUTE SHEET ( RULE 26) are present in the detector having primer polynucleotides that specifically bind to different target genes or target polynucleotides, it is also possible that binding one or more target polynucleotides can reflect binding to different targets.

[0033] The target polynucleotides can come from a variety of organisms. For example, in some embodiments, the target polynucleotide is from a vertebrate organism such as a human. In other embodiments, the target polynucleotide is associated with a disease such as cancer. In other embodiments, the target polynucleotide is obtained from a pathogenic organism such as a virus or bacteria. In some embodiments, the target polynucleotides are viral polynucleotides. For example, the target virus may be, for example, a Zika virus (ZIKV), hepatitis B virus (HBV), hepatitis C virus (HCV), dengue virus (DENV-1 and -2), human cytomegalovirus (HCMV), herpes simplex virus (HSV), etc.

Target Polynucleotide Detection Systems

[0034] Another aspect of the invention provides a system for detecting a target polynucleotide in a sample, the system comprising: a microchip comprising at least one channel configured to receive a sample comprising one or more target polynucleotides, one or more LAMP probes bound to a surface of the channel that specifically bind to the one or more target polynucleotides, a source of detectable nanoparticles that bind to the LAMP probes, and a detector configured to detect a signal created by the detectable nanoparticles.

[0035] The system also includes a mobile device comprising: a camera configured to acquire an image of the signal created by the detectable nanoparticles in the microchip; a neural network configured to receive the acquired image and to generate a probability regarding the presence of the one or more target polynucleotides in the sample based on the acquired image; and a display coupled to the neural network and configured to display the probability regarding the presence of the one or more target polynucleotides in the sample.

[0036] Figure 2 provides a block diagram of a system for polynucleotide detection using nanoparticles and neural network enabled mobile devices. The system 100 includes mobile device 102 and a microchip 104. The microchip 104 is configured to receive a sample 106 (e.g., blood, plasma/serum, serum) to determine if the sample includes the target polynucleotide. The mobile device 102 (e.g., a smartphone, a tablet, etc.) may be configured to acquire an image of the microchip 104 and the sample 106 and to analyze the image using a neural network to

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SUBSTITUTE SHEET ( RULE 26) determine whether the sample 106 is infected or not infected with the target virus based on the bubble formation on the microchip 104.

[0037] The mobile device 102 includes a camera 108, a neural network 110, an output 112 of the neural network 110, a display 114 and a memory 116. The camera 108 may be configured to allow a user of the mobile device 102 to acquire an image of the microchip 104 and sample 106. Advantageously, the image pf the microchip 104 and sample 106 may be directly acquired by the camera 18 of the mobile device 102 without an optical attachment for the mobile device 102.

[0038] The acquired image of the microchip 104 and sample 106 may be input into the neural network 110 which is configured and trained to generate an output 112 indicating whether the sample 106 includes the target polynucleotide (i.e., positive) or does not include the target polynucleotide (i.e., negative) based on the acquired image of the microchip 104 and sample 104. The neural network may be trained using known methods. In some embodiments, the neural network 110 is a convolutional neural network (CNN) such as, for example, an Inception v3 architecture, that may be pre-trained using the ImageNet image database. The pre-trained CNN may then be fine-tuned with a training data set that includes pre- labeled images of bubble formations or patterns on microchips using, for example, various target polynucleotides (e.g., viral polynucleotides), polynucleotide concentrations, and different dilutions of nanoparticles (e.g., platinum nanoparticles (PtNPs)). In some embodiments, the neural network output 112 is a probability value of the sample 106 being positive or negative for the target polynucleotide. The output 112 may be displayed on a display 114 of the mobile device 102. The output 112 may also be stored in the memory 116 of the mobile device 102.

[0039] In some embodiments, the mobile device 102 may be, for example, a mobile phone, a smartphone, a tablet, or the like, or other standalone optical systems for imaging. As such, the mobile device 102 may include any suitable hardware and components designed or capable of carrying out a variety of processing and control tasks, including steps for acquiring an image of the microchip using camera 108, implementing the neural network 110, providing the output 112 to the display or storing the output 112 in memory 116. For example, the mobile device 102 may include a programmable processor or combination of programmable processors, such as central processing units (CPUs), graphics processing units (GPUs), and the like. In some implementations, the mobile device 102 may be configured to execute instructions stored in a

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SUBSTITUTE SHEET ( RULE 26) non-transitory computer readable-media. In this regard, the mobile device 102 may be any device or system designed to integrate a variety of software, hardware, capabilities and functionalities. Alternatively, and by way of particular configurations and programming, the mobile device 102 may include a special-purpose system or device. For instance, such specialpurpose system or device may include one or more dedicated processing units or modules that may be configured (e.g., hardwired, or pre-programmed) to carry out steps, in accordance with aspects of the present disclosure.

[0040] Computer-executable instructions for polynucleotide detection using nanoparticles and a neural network enabled mobile device according to the above-described methods may be stored on a form of computer readable media. Computer readable media includes volatile and nonvolatile, removable, and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data. Computer readable media includes, but is not limited to, random access memory (RAM), read-only memory (ROM), electrically erasable programmable ROM (EEPROM), flash memory or other memory technology, compact disk ROM (CD-ROM), digital volatile disks (DVD) or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired instructions and which may be accessed by a system (e.g., a computer), including by internet or other computer network form of access.

[0041] The system can include any of the embodiments of the device described herein. For example, in additional embodiments, the nanoparticles are metal nanoparticles. In further embodiments, the target polynucleotides are viral polynucleotides.

[0042] The acquired image may be provided to a neural network on the mobile device. In some embodiments, the neural network can be trained to generate an output, for example, a virus detection classification, indicating whether the sample includes the target polynucleotide (i.e., positive) or does not include the target polynucleotide (i.e., negative) based on the acquired image of the microchip and sample. The neural network may be trained using known methods. In some embodiments, the neural network is a convolutional neural network (CNN) such as, for example, an Inception v3 architecture, that may be pre-trained using the ImageNet image database. The pre-trained CNN may then be fine-tuned with a training data set that includes pre-labeled images of bubble formations or patterns on microchips using, for example,

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SUBSTITUTE SHEET ( RULE 26) various target viruses, target virus concentrations, and different dilutions of nanoparticles (e.g., PtNPs). At block, the neural network generates the virus detection classification of the acquired image. For example, in some embodiments, the neural network generates a probability value of the sample being positive or negative for the target polynucleotide (e.g., viral polynucleotide). The output of the neural network (e.g., the generated probability value(s) and the acquired image of the microchip and sample) may be displayed on a display of the mobile device and/or stored in the memory of the mobile device.

[0043] In some embodiments, the neural network of the mobile device (e.g., a smartphone, tablet, etc.) may be a deep learning CNN. A CNN model can be trained to analyze bubbles formed on an image of a single channel microchip with a sample from a subject to qualitatively identify samples as, for example, positive or negative for the target virus as discussed above. In some embodiments, the CNN model may be configured to perform supervised learning to automatically recognize differences between two classes of positive (infected) and negative (non-infected) samples. For example, the CNN model can use the Inception v3 architecture. In some embodiments, the CNN may be pre-trained using the ImageNet image database, for example, a dataset of 1,000 object classes containing 1.28 million images of the 2014 ImageNet Challenge. In an embodiment, transfer learning may then be performed by removing the final classification layer from the CNN and re-training (or fine tuning) the CNN with a training dataset of images of microchips containing bubbles analogous to virus samples. For example, the raining dataset for fine tuning the CNN may include pre-labeled images of single-channel microchips with bubbles (e.g., formed from various target viruses, target virus concentrations, and different dilutions of nanoparticles) and organized in the two different classes (positive and negative) for training. In some embodiments, each image in the training dataset for fine-tuning may be resized (e.g., 299 x 299 pixels) to be compatible with the original dimensions of the Inception v3 network architecture. Transfer learning can leverage the natural-image features learned by the ImageNet pre-trained network. In some embodiments, the CNN may be trained using back propagation and all layers of the network may be fine-tuned using the same global learning rate of 0.001. As discussed above, the CNN may be configured to provide the probability value of the tested sample as being positive or negative.

Methods of Detecting a Biological Analyte

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SUBSTITUTE SHEET ( RULE 26) [0044] Another aspect of the invention provides a method for detecting a target polynucleotide in a sample. The method includes the steps of loading a sample into a microchip comprising at least one channel configured to receive a sample comprising one or more target polynucleotides, one or more LAMP probes bound to a surface of the channel that specifically bind to the one or more target polynucleotides, a source of detectable nanoparticles that bind to the LAMP probes, and a detector configured to detect a signal created by the detectable nanoparticles; binding the one or more target polynucleotide to the one or more LAMP probes; amplification of the one or more target polynucleotide using the one or more LAMP probes; providing detectable nanoparticles and binding them to the LAMP probes; and detecting the detectable nanoparticles in order to detect the one or more target polynucleotides. In some embodiments, the amplification of the one or more target polynucleotides is conducted isothermally.

[0045] In some embodiments, the method further comprises using a mobile device to evaluate and display the detected target polynucleotides. The mobile device comprises: a camera configured to acquire an image of the signal created by the detectable nanoparticles in the microchip; a neural network configured to receive the acquired image and to generate a probability regarding the presence of the one or more target polynucleotides in the sample based on the acquired image; and a display coupled to the neural network and configured to display the probability regarding the presence of the one or more target polynucleotides in the sample.

[0046] The method can include any of the embodiments of the device described herein. For example, in some embodiments, the target polynucleotides are viral polynucleotides. The LAMP reaction mixture consists of dNTPs mix, Bst polymerase, fluorescence dye, primers and DNA template. When different LAMP probes are used to detect different target polynucleotides, the different LAMP probes can be segregated to different locations in the device to allow the detection of the different target polynucleotides to be distinguished from one another.

[0047] In some embodiments of the method, the detectable nanoparticles comprise platinum capable of catalyzing oxygen gas formation from hydrogen peroxide, and wherein the step of detecting the detectable nanoparticles further comprises providing a hydrogel peroxide solution to the channel of the microchip.

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SUBSTITUTE SHEET ( RULE 26) [0048] In some embodiments, the method includes the step of obtaining a biological sample from a subject and passing the biological sample through the flow region of a microfluidic capture device. Biological samples include, but are not necessarily limited to bodily fluids such as urine and blood-related samples (e.g., whole blood, serum, plasma, and other blood- derived samples), urine, cerebral spinal fluid, bronchoalveolar lavage, and the like.

[0049] A biological sample may be fresh or stored (e.g. blood or blood fraction stored in a blood bank). Samples can be stored for varying amounts of time, such as being stored for an hour, a day, a week, a month, or more than a month. The biological sample may be a bodily fluid expressly obtained for use in the microfluidic device of this invention or a bodily fluid obtained for another purpose which can be subsampled for the assays of this invention. In some embodiments, it may be preferable to filter, centrifuge, or otherwise pre-treat the biological sample to remove impurities or other undesirable matter that could interfere with analysis of the biological sample.

[0050] An example has been included to more clearly describe particular embodiments of the invention. However, there are a wide variety of other embodiments within the scope of the present invention, which should not be limited to the particular example provided herein.

EXAMPLE

Example 1 : LAMP testing

[0051] This technology relies on using streptavidin (SA)-functionalized microchip to specifically capture biotinylated- HIV DNA amplicons and SA-functionalized PtNPs (i.e., Pt- nanoprobes) to label the LAMP amplicons captured on the surface of chip, inducing the formation of bubbles in the presence of peroxide solution, as shown in Figure 3.

[0052] Following this design, we successfully developed and tested the two major components of this technology: (i) SA-functionalized microchip that specifically captures biotinylated-HIV LAMP DNA amplicons, and (ii) SA-functionalized PtNPs that can label the LAMP amplicons and form catalytically active 3D nanoparticle-DNA structures to induce the formation of gas bubbles on-chip. In addition, we successfully tested the developed microchip and nanoparticle systems for the detection of a model target nucleic acid of HIV-1 RNA with a detection limit of 100 copies/ml.

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SUBSTITUTE SHEET ( RULE 26) [0053] The SA-functionalized microchips with a single microfluidic channel were prepared using a 3mm laser-machined poly(methyl methacrylate) (PMMA) (3.175 mm; McMaster-Carr, 8560K239) and double-sided adhesive (DSA) (80 mm; 3M, 82603) assembled on a glass substrate (25 x 75 mm; Globe Scientific Inc.) (Figure 3). The glass surface of the microchip was initially salinized with triethoxysilane-polyethylene-glycol-thiol (Si-PEG-SH) (Nanocs Inc., PG2-SLTH-5k), and the free thiol (-SH) groups exposed on the surface of the microchip were then allowed to react with maleimide-activated streptavidin (Nanocs Inc., SV1-ML-1) by thiol-maleimide reaction. The functionalization of the surface of the chip was confirmed using FT-IR and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques. FT-IR analysis showed that the streptavidin- functionalized microchip and the antibody-functionalized chip (as a positive control chip) displayed many bands: the absorption bands of amide I groups at 1735 cm’ ¹ (C-0 stretching vibration of peptide linkages), and the amide II groups around 1592 cm’ ¹ , providing proof of the successful surface modification of the chip. In addition, peaks at 922.8 cm ¹ , 721.4 cm’ ¹ , 650 cm ¹ , and 560.9 cm’ ¹ are attributable to Si-O-Si stretching, H-Si-0 vibrations, and C-H that reflect the efficient salinization and pegylation of the surface of the chip. The SDS-PAGE pattern of streptavidin- functionalized microchip had only one protein band representing streptavidin subunits around 18 kDa. The results for antibody-functionalized microchips showed the presence of two major protein bands around 50 kDa and 25 kDa, which are characteristic of IgG heavy and light chains, respectively.

[0054] The Pt-nanoprobes were prepared from a direct conjugation of thiol-activated SA to the surface of citrate PtNPs, by the well-known thiol-metal bond (Figures 4A-4C). Polsky, R., et al., Analytical Chemistry, 78(7): p. 2268-2271 (2006); Draz, M.S., et al., Nature communications, 2018. 9(1): p. 1-13 (2018); Draz, M.S. et al., ACS nano, 12(6): p. 5709-5718 (2018). Transmission electron microscopy (TEM) and the corresponding size distribution histogram show that the synthesized PtNPs are spherical with an average diameter of 11.3+1.2 nm. The efficiency of the coupling reaction of SA and antibody to the surface of PtNPs was assessed by agarose gel electrophoresis, UV-vis spectroscopy, and Fourier transform infrared spectroscopy (FT-IR) techniques. Agarose gel electrophoresis showed that the migration of PtNPs is slightly retarded post-conjugation to SA compared with unmodified PtNPs, suggesting the addition of SA molecules to their surface, which partially interferes with electrophoretic behavior due to the different size and charge density value between PtNPs, and the SA-PtNPs conjugates (i.e., Pt-nanoprobes).

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SUBSTITUTE SHEET ( RULE 26) [0055] Using the functionalized microchip and Pt-nanoprobe systems, we successfully tested our protocol for nucleic amplification and bubble formation on a chip. The HIV-1 RNA in each of the tested samples was amplified using the LAMP protocol. The reaction was performed as follows: a mixture of DNA primers (35 pM) was prepared then added to the reaction mixture, which is prepared from 2.5 pl isothermal amplification buffer (New England Biolabs Inc., BO537S), 1.5 pl MgSO ₄ (100 mM), 2 pl dNTP (25 mM), and 3 pl Betaine (5 M). Then 5 pl of the target RNA was added, followed by adding 5 units of AMV reverse transcription enzyme (New England Biolabs Inc., M0277L), and 10 units of Bst 2.0 DNA Polymerase (New England Biolabs Inc., M0537L). The reaction volume was brought to 20 pl by UltraPure™ DNase/RNase-Free Distilled Water (Thermo Fisher Scientific Inc., 10977023) and mixed thoroughly before incubation for 20 minutes at 70°C and termination at 85°C for 5 minutes. The formed biotinylated LAMP DNA amplicons were loaded onto the surface of the streptavidin-modified microchip for DNA capturing. The captured DNA amplicons were then labeled with Pt-nanoprobes (prepared with streptavidin) for bubble formation in the presence of a peroxide solution (10% H2O2-20% glycerol-70% DI water).

[0056] A variety of methods were used to confirm formation of the platinum complexes. See Figure 5. Agarose gel electrophoresis patterns of the amplification products of HIV-1 RNA confirmed the formation of large multi-banded DNA amplicons (Figure 5C). The efficiency of DNA capturing and bubble formation on the surface of the chip was evaluated using UV-vis absorbance, fluorescence spectroscopy, and bright-field light microscopy. The results of UV- vis absorbance showed that up to 96.23% of the added LAMP DNA was captured on the surface of the microchip. Fluorescence spectroscopy indicated the successful capturing of the formed DNA amplicons on the surface of the microchip and a direct increase in the fluorescence signal with an increased concentration of the added DNA amplicons to the chip. The formed bubbles on the chip were imaged and analyzed using bright-field light microscopy. The size and number of bubbles were directly proportional to the incubation time of the chip (after the addition of H2O2 solution). The average size of the bubbles was 249.65 ± 39.96 pm after 180 s.

[0057] In addition, we successfully tested PBS samples (N=50) spiked with different concentrations of the target HIV-1 (from undetectable to 10 ⁹ copies/ml) and non-targeted competing viruses including HCV, HBV, and HPV-16/18 (at 10 ⁹ copies/ml). The HIV-1 RNA in each of the tested samples was amplified using the LAMP protocol. The formed amplicons were loaded on-chip, incubated for 10 minutes, then labeled with the Pt-nanoprobes for 5

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SUBSTITUTE SHEET ( RULE 26) minutes, and the generated bubbles were detected using a cellphone system empowered with an Al-based algorithm. The Al algorithm was trained and tested to detect and differentiate the features of chips with concentrations above and below a threshold of 100 copies/ml for HIV-1 RNA (the threshold of highly sensitive assays for HIV-1 detection). Swenson, L.C., et al., Journal of clinical microbiology, 52(2): p. 517-523 (2014). The results indicated a direct increase of the bubble number with the increase of the tested HIV-1 RNA concentration, and no bubbles were observed at control samples (no RNA was added). Furthermore, the cellphone system showed a 100% detection accuracy in the qualitative detection of HIV-1 RNA-spiked PBS samples and a good agreement with the tested HIV-1 RNA concentrations (Figure 6).

[0058] We tested the effect of the presence of nonbiotinylated DNA amplicons and biotin spiked in a complex biological sample such as blood at a relatively high concentration of 10 pg/ml. We have not observed interference from biotin (only small bubbles were formed after prolonged incubation with H2O2 for 20 minutes) or other inhibitory factors in blood samples (Figure 7). Bubble formation was confirmed as shown in Figure 7B.

[0059] In conclusion, we developed an inexpensive platform technology for nucleic acid testing that is sensitive, accurate and reliable, and easy-to-use to allow for rapid nucleic acid testing. This technology will enable (i) disease management at-home, (ii) wider and more flexible testing, (ii) timely initiation and monitoring of treatments, (iii) detection of treatment failure, (iv) evaluating new strategies for disease treatment, and (v) testing of drug resistance. We, therefore, anticipate that our research will have a major impact on disease testing, turn- around-time, epidemiology and incidence of diseases, and adherence to care and treatment.

[0060] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

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SUBSTITUTE SHEET ( RULE 26) CLAIMS

What is claimed is:

1. A device for detecting a target polynucleotide in a sample, comprising a microchip comprising at least one channel configured to receive a sample comprising one or more target polynucleotides, one or more loop-mediated isothermal amplification (LAMP) probes bound to a surface of the channel that specifically bind to one or more target polynucleotides, a source of detectable nanoparticles that bind to the LAMP probes, and a detector configured to detect a signal created by the detectable nanoparticles.

2. The device of claim 1, wherein the nanoparticles are metal nanoparticles.

3. The device of claim 2, wherein the metal nanoparticles comprise platinum capable of catalyzing oxygen gas formation from hydrogen peroxide.

4. The device of claim 1, wherein the target polynucleotides are viral polynucleotides.

5. A system for detecting a target polynucleotide in a sample, the system comprising: a microchip comprising at least one channel configured to receive a sample comprising one or more target polynucleotides, one or more LAMP probes bound to a surface of the channel that specifically bind to one or more target polynucleotides, a source of detectable nanoparticles that bind to the LAMP probes, and a detector configured to detect a signal created by the detectable nanoparticles; and a mobile device comprising: a camera configured to acquire an image of the signal created by the detectable nanoparticles in the microchip; a neural network configured to receive the acquired image and to generate a probability regarding the presence of the target polynucleotide in the sample based on the acquired image; and a display coupled to the neural network and configured to display the probability regarding the presence of the target polynucleotide in the sample.

6. The system of claim 5, wherein the nanoparticles are metal nanoparticles.

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SUBSTITUTE SHEET ( RULE 26) 7. The system of claim 5, wherein the target polynucleotides are viral polynucleotides.

8. The system of claim 5, wherein the mobile device is a smartphone.

9. The system of claim 5, wherein the neural network is a convolutional neural network.

10. A method for detecting a target polynucleotide in a sample, comprising: loading a sample into a microchip comprising at least one channel configured to receive a sample comprising one or more target polynucleotides, one or more LAMP probes bound to a surface of the channel that specifically bind to one or more target polynucleotides, a source of detectable nanoparticles that bind to the LAMP probes, and a detector configured to detect a signal created by the detectable nanoparticles; binding the one or more target polynucleotides to the one or more LAMP probes; amplification of the one or more target polynucleotides using the one or more LAMP probes; providing detectable nanoparticles and binding them to the LAMP probes; detecting the detectable nanoparticles in order to detect the one or more target polynucleotides.

11. The method of claim 10, wherein the sample is obtained from a subject.

12. The method of claim 10, wherein the amplification of the one or more target polynucleotides is conducted isothermally.

13. The method of claim 10, wherein the method further comprises using a mobile device to evaluate and display the detected one or more target polynucleotides, the mobile device comprising: a camera configured to acquire an image of the signal created by the detectable nanoparticles in the microchip; a neural network configured to receive the acquired image and to generate a probability regarding the presence of the target polynucleotide in the sample based on the acquired image; and a display coupled to the neural network and configured to display the probability regarding the presence of the target polynucleotide in the sample.

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SUBSTITUTE SHEET ( RULE 26) 14. The method of claim 13, wherein the target polynucleotides are viral polynucleotides.

15. The method of claim 13, wherein the detectable nanoparticles comprise platinum capable of catalyzing oxygen gas formation from hydrogen peroxide, and wherein the step of detecting the detectable nanoparticles further comprises providing a hydrogel peroxide solution to the channel of the microchip.

SUBSTITUTE SHEET ( RULE 26)