Integrated pore-based detection for lateral flow nucleic acid assay

Abstract

This invention provides an apparatus for lateral flow nucleic acid assays and a method for using the same. [Solution] An apparatus having an integrated pore-based detector capable of detecting both microbial and viral pathogens in aqueous samples within approximately 5 minutes without nucleic acid amplification or optical components. The detector is based on an electromechanical signaling mechanism that enables low-cost detection of DNA / RNA at ultra-low concentrations (up to approximately 10 M to approximately 19 M). This scheme relies on the use of charged neutral peptide nucleic acid (PNA) capture probes conjugated to polystyrene beads. The PNA beads acquire a substantial negative charge upon capture of target pathogenic DNA / RNA, making them mobile in an electric field. When a bias voltage of approximately 1 V to 2 V is applied, the PNA beads with the hybridized target are electrophoretically guided into smaller diameter pores. Subsequent pore blockade results in a strong and sustained decrease in the measured ion current through the pores.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is U.S. Provisional Patent Application No. 63 / 075,669, filed on September 8, 2020. The prior and interest of the said, which is incorporated herein by reference in its entirety.

[0002] Description of funding provided by the federal government. Research or development Not applicable

[0003] Notice of copyrighted material Some of the materials in this patent document are protected by copyright under the copyright laws of the United States and other countries. It is possible. The copyright holder may not be found in any publicly available files or records of the United States Patent and Trademark Office. Therefore, I will not object to reproduction in either the patent document or the patent disclosure, but otherwise All copyrights are reserved. The copyright holders are entitled to rights in accordance with 37 CFR §1.14. We waive any right to keep this patent document confidential, including but not limited to the above. do not have.

[0004] 1. Technical field

[0005] The technology of this disclosure generally involves complementary conjugated charged neutral polystyrene beads Regarding the detection of specific RNA or DNA fragments using probes, more specifically, charge-neutral probes Specific RNA or DN using complementary probes conjugated on lithyrene beads Regarding the detection of transverse flow in A-fragments. [Background technology]

[0006] 2. Background Explanation Low-cost, accurate, and robust point-of-care (POC) nucleic acids that deliver results in minutes. The conception and development of NA-based diagnostic devices are being strongly promoted. Currently, most infectious diseases Diagnosis is typically achieved by a culture method that takes several days. POC immunoassay is used to detect diseases. Although commercially available for detecting the active ingredient, these have limited sensitivity and specificity. Nucleic acid (NA)-based tests, while numerous, are not very common and have extremely low limit of detection (LOD). Low sensitivity and specificity, ranging from 90% to 99%. Influenza, respiratory syncytia For a small number of analytes containing somatic virus (RSV) and Group A Streptococcus, a small number of POCs were used. NA-based testing is available.

[0007] NA-based tests for other indications include Neisseria gonorea. gonorrhea (NG, gonorrhea) and Chlamydia trachomatis (Chla Clinical laboratory for cases such as mydia trachomatis (CT, chlamydia) It is a test, and the process of transporting samples to the laboratory, batch processing, testing, and returning results is also involved. Typically, this takes several days. The key points regarding the significance of the technology disclosed here are summarized below.

[0008] Most current methods for diagnosing infectious diseases take more than a day, which means that optimal treatment and diagnosis are not possible. Unreliable follow-up contact may hinder the rapid administration of counseling. This leads to dependence on antibiotics, inappropriate prescriptions of antibiotics, and prolonged patient suffering. This could potentially contribute to high medical costs.

[0009] Rapid determination of the presence or absence of important pathogens in clinical samples, i.e., qualitative testing. The tests typically detect influenza, RSV, SARS-CoV-2, HIV, and human papillomavirus (HPV). The most important need includes viruses (HPV), NG, CT, etc.

[0010] Methods with sufficiently low detection limits based on NA detection without amplification are rare and generally require expensive reagents and / or complex analytical devices.

[0011] Determining the simple presence or absence (yes / no answer) of important pathogens in body fluids, that is, qualitative tests are the most important need. The mere presence of it at any level in body fluids is abnormal and common pathogens that are indicators of infectious diseases include SARS-CoV-2, group A Streptococcus (Streptococcus), Neisseria gonorrhoeae (NG: Neisseria gonorrhoeae), Chlamydia trachomatis (CT: Chlamydia trachomatis), influenza virus, and Bordetella pertussis (whooping cough). is included.

[0012] Pathogen detection methods based on NA amplification have important drawbacks. All NA amplification-dependent devices must include subsystems for sample preparation (including pathogen lysis and NA purification), NA target amplification, and amplicon detection.

[0013] <~ Generally, optical methods are used for amplicon detection, which requires the incorporation of optical components into the device related to increased complexity and cost. Despite remarkable progress in rapid methods for polymerase chain reaction (PCR) cycling, due to the need for accurate temperature control, many test developers have come to pursue isothermal amplification methods, but these methods still require the removal of primers, polymerase, and polymerase inhibitors. ​​Require extensive NA purification and reaction conditions that must be carefully controlled.

[0014] Broadly applicable, NA amplification-free, label-free, sequence-specific NA detection schemes are rare. In the past decade or so, there has been no significant progress in the development of new approaches for amplification-free NA detection at clinically relevant concentrations below the single-digit attomolar (aM, 10 -18 M) range. However, only a handful of these schemes do not require special labels other than oligonucleotides complementary to the target NA. Also, nearly half require some kind of optical system. The remaining approaches involve piezoelectric, MALDI TOF MS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometry), or various electrochemical techniques. Among these schemes based on electrochemical detection, only one uses simple and inexpensive constant-potential amperometry, but still requires Pt nanoparticle labels. Ideally, an NA sensor without amplification would only contain selective oligonucleotide probes and would eliminate the need for additional reagents, labels, or complex signal transduction techniques. However, the state-of-the-art presented above suggests that detection methods that meet this ideal are rare.

[0015] Of these schemes based on electrochemical detection, only one uses simple and inexpensive constant-potential amperometry, but still requires Pt nanoparticle labels. Ideally, an NA sensor without amplification would only contain selective oligonucleotide probes and would eliminate the need for additional reagents, labels, or complex signal transduction techniques. However, the state-of-the-art presented above suggests that detection methods that meet this ideal are rare. The state-of-the-art presented above suggests that detection methods that meet this ideal are rare.

[0016] Previous studies

[0017] The RNA / DNA detection device disclosed herein monitors the conductance of pores or channels filled with electrolyte when various analytes cross it. Coulter ( DeBlois RW,Bean CP.Counting and Sizing o​​​​​​​​ f Submicron Particles by the Resistive P ulse Technique.Review of Scientific Instruments Resistive pulse sensor based on the study (Ruments. 1970;41(7):909-16) It differs from other nanopore-based NA detection systems in that it is not a sa. Rather, this This is based on a much simpler conductivity measurement detection of large signals from pore blockage that persist for a long time. It is.

[0018] The resistance pulse technique is used to analyze the analyte on a short timescale (μs~ms) as it passes through the pores. While the focus is on the precise measurement of small changes in nanopore currents, the details disclosed herein are The instrument technology essentially amplifies this signal to the nA range and has the ability to indicate the presence of the analyte. By relying on continuous pore blockage, its duration is extended indefinitely.

[0019] This method was published on March 7, 2013, and is incorporated herein by reference in its entirety. Born from Monbouquette, Harold and Schmidt, Jacob, P As described in CT International Publication No. 2013 / 033647, Deba Significantly simplifies the operation of electronic devices and reading data.

[0020] Nonspecifically bound NA provides little to no persistent signal. "Signal" is, Note that this is a sustained, gradual decrease in ionic current lasting for several seconds or more. Non-complementary NA Most control executions do not result in observable pore blockade, but rather some transient blockade. Only interruptions (not lasting long enough to constitute a signal) are rarely observed. However, non-complementary signals are present. Incubation of beads with target NA involves a range from single digits to approximately 20-30mV. As indicated by the increase in potential, it can occasionally result in substantial nonspecific binding. It is suspected that these beads, which have nonspecifically bound DNA, are negatively affected. It becomes charged, electrophoretically mobile, and can be driven into pores.

[0021] At the pore opening, the electric field is strong enough to remove DNA nonspecifically bound to the beads. This causes a decrease in bead charge and electrophoretic mobility, and the opposite direction of electroosmotic flow. The drag force allows the beads to be carried out of the pores by the electrophoretic force. This electroosmotic flow This arises from the counterflow of opposing ions against a fixed negative charge on the glass pore wall.

[0022] Experimental evidence, as well as the results of many controlled studies, shows that only nonspecifically bound NA can be found in The particles briefly approach the pore opening, and then, despite the possibility of mobility due to dielectrophoresis... This shows that it is swept away by the opposite electroosmotic flow. The literature shows that to avoid false positives I don't think they've disclosed another NA-based diagnostic system with such an active system. It breaks. [Prior art documents] [Patent Documents]

[0023] [Patent Document 1] International Publication No. 2013 / 033647 [Non-patent literature]

[0024] [Non-Patent Document 1] DeBlois RW,Bean CP.Counting and Sizing of Submicron Particles by the Resistive Pulse Technique.Review of Scientific Instruments.1970;41(7):909-16 [Overview of the project]

[0025] This technology enables lateral flow nucleic acid assays using an integrated pore-based detector. Instructions for using Biso are provided below.

[0026] In one embodiment, the technology described herein relates to a thin glass film and a glass having pores. Integrating the top with the lateral flow membrane, and magnetic polystyrene beads-PNA (pe Using a butylated nucleic acid conjugate, we control the position of the beads on the membrane and hybridize them. The target nucleic acid was used to detect the bead-PNA conjugate by glass chipping the beads. This includes arranging in close proximity to the . In this embodiment, magnetic polystyrene beads-PNA are Although used, it contains other charged neutral nucleic acid analogs, including charged neutral peptide nucleic acid (PNA) capture agents. It should be noted that other magnetic substrates that can be conjugated with the capture probe can also be used. It should be done.

[0027] The integrated device can analyze nucleic acid in aqueous samples within approximately 5 minutes without nucleic acid amplification or optical components. It has the potential to detect both microbial and viral pathogens. The detector can detect ultra-low concentrations (10 - 19 Low-cost, optical-free, and amplification-free analysis of DNA / RNA at low concentrations (e.g., P It relies on a novel electromechanical signaling mechanism that enables detection (without CR).

[0028] A key feature of the detector is its ability to detect uncharged polyamide analogs for NA, which share the same base chemistry. This involves the use of peptide nucleic acid (PNA) capture probes. Bead-PNA conjugate Since they are designed to be charge-neutral, they cannot be sensed in the presence of a DC electric field. It does not exhibit sufficient electrophoretic motion. However, the substantial amount obtained when capturing the target NA sequence A typical negative charge makes the hybridized conjugate movable.

[0029] Smaller direct current of bead-PNA conjugates with hybridized target NA Electrophoresis of glass pores of a certain diameter causes a significant increase in pore resistance, thereby, This results in a persistently strong, sustained decrease in the measured ion current. Nonspecifically bound N A is removed from the bead conjugate by the strong electric field inside the pore opening, resulting in a sustained signal. Furthermore, the opposite electroosmotic flow through the glass pores does not result in high osmotic flow away from the pore opening. Sweep the PNA-bead conjugate without breeding targets. In this way, This simple conductivity measuring device signals the presence or absence of target NA (and associated pathogens). It transmits a nal signal, giving a highly selective (false positive, rarely observed) binary response.

[0030] The diagnostic applications of the apparatus and methods include, but are not limited to, the following: 1. Any microorganism or These are viral pathogens, such as SARS-CoV-2, influenza, gonorrhea, and chlamydia. 1. Use in clinics, emergency rooms, or emergency treatment centers; 2. CO VID-19 screening, e.g., dental prostheses, surgical appointments, on-site, small meetings; 4. Home use Diagnosis; 5. Food safety; and 6. Foot-and-mouth disease (cattle).

[0031] Further military diagnostic applications may include, but are not limited to, the following: diarrheal diseases; infected wounds. Partial assays; biological weapons agents, e.g., anthrax, plague; and site-specific pathogens, e.g., densitic malformations. Yellow fever.

[0032] The apparatus and method are robust, low-power (e.g., battery-powered), and possibly handheld. Yes, and it is rapid (less than 5 minutes for detection).

[0033] Further aspects of the technology described herein are revealed in the following sections of this specification. The description aims to fully disclose preferred embodiments of the technology without imposing limitations. ru.

[0034] The technology described herein will be better understood by referring to the following drawings. Yes, these drawings are not to scale and are for illustrative purposes only. [Brief explanation of the drawing]

[0035] [Figure 1] This is an overall diagram of a detection scheme for specific nucleic acids using PNA probe conjugate charged neutral polystyrene beads. [Figure 2A] This is a photograph of a 1 cm square borosilicate glass sample with a submicron-thick film, micromachined, in the center. [Figure 2B] This is a scanning electron microscope (SEM) image of the etched nanopore film shown in Figure 2A, viewed from an oblique angle. [Figure 2C] Figure 2B shows a focused ion beam (FIB)-etched nanopore SEM of an etched film, which can be used as a pore for nucleic acid detection as described herein. [Figure 3A] This is a side view of a lateral flow nucleic acid assay with integrated pore-based detection. [Figure 3B]This is an enlarged cross-section of Figure 3A, which more clearly shows the geometric shape of the pores used for detection in the integrated pore base. [Figure 3C] This is a top view of the polydimethylsiloxane (PDMS) upper pattern deposited on a glass chip. [Figure 4] This is a diagram of an apparatus for detecting specific nucleic acids using probe-conjugate charged neutral polystyrene beads. [Figure 5A] This diagram provides an overall flowchart of a method for detecting specific nucleic acids using probe-conjugate charged neutral polystyrene beads. [Figure 5B] This diagram provides an overall flowchart of a method for detecting specific nucleic acids using probe-conjugate charged neutral polystyrene beads. [Figure 6] This is a side view of a lateral flow zone assembly. [Figure 7A] This is a side view of a glass chip assembly. [Figure 7B] This is a top view of the polydimethylsiloxane (PDMS) upper pattern deposited on a glass chip. [Figure 7C] This is a top view of a polydimethylsiloxane (PDMS) bottom film deposited on a glass chip. [Figure 8] This is a side view of the entire system assembly. [Figure 9] This is a plot of pore currents observed using a potentiostat to measure the ion current passing through the pores while the potential is fixed. [Modes for carrying out the invention]

[0036] Here, we refer to Figure 1, which is a diagram 100 of the operating characteristics of this device. First, the film 102 Positive voltage V + 104 and negative voltage V - It is positioned between 106 and 106.

[0037] This diagram shows one or more complementary elements to a single-stranded nucleic acid target (DNA or RNA, 112). Polystyrene beads 108 having a covalent peptide nucleic acid (PNA) probe 110, and This shows pores 114 passing through a glass film 102 with a diameter smaller than that of beads 108. 8 is a surface calc used as a binding site for the amine-terminated PNA probe 110. It is purchased together with a boxyl group. A particular bead 108 discussed in one embodiment has a diameter of 8 It is 20nm.

[0038] Other beads and pore dimensions, as well as geometric shapes, can function well. Submicron thickness The film performed best, but if other materials are used, good performance is limited to this dimension. It was found that it was not present. Furthermore, although the target of the manufacturing was a cylindrical pore 114, it was essentially circular. A conical shape was obtained. The minimum size of the pores 114 was sufficiently small, and one or more hybrids The soybean beads block the ion current passing through the pores 114, resulting in a decrease in the pore current 114. Other pore shapes can also function as long as they are possible.

[0039] First, a specific single-stranded nucleic acid target (DNA or RNA, 112) is dissolved as an unbound portion. It is encapsulated in liquid. However, after a while, it targets specific single-stranded nucleic acid targets (DNA or RNA, 112) has one or more covalent peptide nucleic acid (PNA) probes 110 that are complementary to 112). The polystyrene beads 108 achieve hybridization with their targets. This is because the covalently bonded peptide nucleic acid (PNA) probe 118 may sometimes be a single strand. Polystyrene beads bound to nucleic acid targets (DNA or RNA, 112) It is indicated in one or more places in 116.

[0040] In this diagram in Figure 1, three polystyrene beads 116 are used to target single-stranded nucleic acid targets (DNA or It can be seen that it hybridized to RNA (112). This binding corresponds to the length of the target. This creates a network of many negative charges on the bonded polystyrene beads 116, thereby applying The positive voltage V + 104 and negative voltage V - Due to the electric field applied between 106, it becomes electric. This enables electrophoretic (or electrokinetic) movement.

[0041] Typically, charged single-stranded nucleic acid targets (DNA or RNA, 112) have much larger pores 114. Therefore, it proceeds without interruption through the pores 114 of the membrane 102. The charged single-stranded nucleic acid target (DNA or RNA, 122) allows the ion current passing through pore 114 to It passes through the much larger pores 114 without disturbing them to a visible degree.

[0042] In this diagram, the single-stranded nucleic acid target (DNA or RNA, 124) is located in the pores 114 of the membrane 102. It has already passed that point.

[0043] PNA is an uncharged nucleic acid analog. The remaining carboxyl group is initially amine-terminated poly The caps are first coated with ethylene glycol (PEG), and then with ethanolamine. It is important that the PNA is conjugated onto the beads at the optimal surface density. The remaining carboxyl groups on the surface must be capped. Here, polyethylene Recall (PEG) is used to help prevent bead aggregation. Ethanolamine It is used to sequester the remaining carboxyl groups and achieve near electrical neutrality. This is necessary. After these bead modification steps, the beads have a zeta potential of a single-digit low negative mV and are essentially neutral and do not move appreciably in a moderate electric field.

[0044] However, RNA and DNA 112 have a significant negative charge, and when the target RNA or DN A hybridizes to the PNA on the modified beads 120, as shown in binding polystyrene beads 11 6, the complex has a negative charge sufficient to move in the applied electric field ( V - 106~V + 104).

[0045] When the PNA beads with hybridized targets that result in binding polystyrene beads 116 approach the opening of the pore 114, a significant and continuous deflection of the ion current occurs. This continuous decrease in the ion current is called "persistence." Here, referring to FIGS. 2A to 2C, all of these are prior art obtained from Koo B, Yorita A

[0046] M, Schmidt JJ, Monbouquette HG. "Amplificat ion-free, sequence-specific 16S rRNA dete ction at 1 aM." Lab Chip. 018;18(15):2291- 9.doi:10.1039 / C8LC00452H. FIG. 2A is a photograph of a 1 cm square borosilicate glass sample with a nanofabricated nanopore microfabricated at the center of the thinly etched region.

[0047] FIG. 2B is a scanning electron image of the etched membrane of the nanopore of FIG. 2A when viewed obliquely.

[0048] image of the etched membrane of the nanopore of FIG. 2A when viewed obliquely. This is a microscope image (SEM).

[0049] Figure 2C shows the etched film in Figure 2B, which was generated by focused ion beam (FIB). This is a SEM of a nanopore. Such a nanopore can be used as pore 114 in Figure 1. stomach.

[0050] Here, borosilicate glass has been used due to its wide range of applications in the scientific community. However, as a rule, porous materials help prevent false positive test results. A fixed negative charge with a substantial surface concentration is available so that gas permeation flow can be generated. It can be made from such materials. Furthermore, composite materials of two or more materials can also be used.

[0051] Refer to Figures 3A, 3B, and 3C here.

[0052] Figure 3A shows a lateral flow nucleic acid assay with integrated pore-based detection. This is a side view 300 of the form. The substrate 302 is a borosilicate glass microscope slide, etc. A glass substrate is used. A film having a bottom surface 306 and a top surface 308 is placed on the glass substrate 302. 304 is positioned. A sample loading area 310 is located on one side of the membrane 304. ru.

[0053] The glass chip 312 has micro or nanopores 114 manufactured as described above in Figure 1. This includes [something]. This pore 114 is difficult to see in this diagram because its diameter is approximately 500 nm. The lass tip 312 is attached to the film 304 at its upper surface 308, and droplets of conductive buffer 316 are placed on it. It is electrically coupled to the platinum electrode 314 on the other side using this method.

[0054] These glass chips 312 are disposable assemblies containing PNA beads and process fluid. It will be incorporated into the cartridge.

[0055] Between the film 304, the bottom surface 306, and the substrate 302, there is also a white wire 320 attached. Gold foil electrode 318 is positioned. Platinum foil electrode 318 and platinum electrode 314 allow ions to enter the pores 1 It is positioned to conduct the sensing current to the glass chip 312 when passing through 14.

[0056] Here, foil electrodes 318 are shown, but simple wires, patterned wires, and Furthermore, other electrode configurations, such as thin-film conductors directly deposited on the substrate 302, can also be used as substitutes. ru.

[0057] During operation, the sample is loaded into the sample loading area 310, and the membrane 304 Through the capillary action of the membrane 304, more importantly, in close proximity to the pores 114, The sample is transported laterally across the tip 312. The membrane 304 is nitrocellulose-based, glass fiber-based, or used in the implementation of the present invention It may be any other material that is essentially inactive with respect to the material.

[0058] An example of membrane 304 is the Fusion5 membrane product from Cytiva (both sides are water permeable). (Not backed as such). Such film 304 is magnetic or non-magnetic PNA beads Having an effective pore diameter large enough for any of them to move through it In the Fusion5 film 304, a separate sample loading region 310 (with different materials) is used. (The configuration) is not necessary, but a separate sample pad can be used. If a liquid sample is used, an additional absorption pad can be added downstream of the glass detector. It can absorb excess liquid, thereby promoting flow along the lateral flow membrane. .

[0059] Figure 3B shows the lateral flow nucleic acid assay with integrated pore-based detection as shown in Figure 3A. This is an enlarged cross-sectional view of the side view. This shows the fine details of the glass chip 312 and the pores 114. It is enlarged so that it can be better recognized.

[0060] Figure 3C shows polydimethylsiloxane (PDMS) deposited on glass chip 312. This is a top view of the pattern.

[0061] Refer to Figures 3A, 3B, and 3C here. Microfabricated nanopore glass chip 31 2 is a more direct interface to high-throughput manufacturing and POC microfluidic devices. To facilitate the manufacture of faces and low-cost devices. Such glass chips 312 Submicron-thick broom with 114 pores ranging from 100 nanometers to micron scale. We use a MEMS (Micro-Electro-Mechanical Systems) process to fabricate yate glass films. It was released.

[0062] Cartridges containing such glass chips 312 are used in inexpensive electronic devices and displays. , and are likely to be inserted into handheld base units, including wireless communication.

[0063] Another practical application of a lateral flow nucleic acid assay with an integrated pore-based detector 300 In this application, magnets 322 are used to maintain the position of the polystyrene beads containing magnetite. Therefore, it has ferromagnetic properties and is therefore attracted to magnet 322. Please note that other magnetic materials may be used to ferromagnetize the Chilen beads. The magnet 322, which may be a neodymium magnet, another permanent magnet, or an electromagnet, is a hybridized magnet. While the sample is being drawn onto the beads to perform the test, the magnetic PNA beads are fixed in place. Used to hold.

[0064] In Figure 3C, the upper pattern 324 of polydimethylsiloxane (PDMS) can be seen. It should be noted that this pattern is when droplets of conductive buffer 316 fall onto the platinum electrode 31 To better prevent spreading away from 4, it is deposited on the upper surface of the glass chip 312. This is better achieved through the circular opening 326 located above the pore 114.

[0065] The target nucleic acid is introduced into the sample loading region 310, and the sample is then placed on the PNA beads. As it flows, it hybridizes to PNA beads. Next, the hybridized target has The beads move toward the glass chip so that they can block the pores 114. The magnet 322 is removed. Typically, the sample is "washed" towards the target pore 114. The transfer occurs through the capillary action of the membrane 304 due to the addition of a chaser fluid that acts to "flow". To be moved.

[0066] Consideration

[0067] This technology is low-cost, low-power (e.g., battery-powered), portable, small, fast, and robust. This represents a potentially significant advance in NA detection, enabling robust devices as part of the device. The detector performs sample collection, cell lysis, NA extraction, and PNA probes on magnetic beads. Ideally integrated with the overall process flow for targeted NA hybridization. It can be done.

[0068] Sample collection and lysis are performed simultaneously in a syringe pre-loaded with lysis buffer. It is highly likely that these will be done separately. The sample (e.g., urine, blood) is drawn into a syringe, Dissolves in about 1 minute (i.e., chemical destruction of the microbial cell envelope or viral capsid) Destruction occurs. Then, a few drops of the dissolved sample are placed in a syringe attached to a sub Sample pad of assay device passed through a micron filter (pore size approximately 0.1 μm) It is deposited on the region. The filter causes pore blockage when negatively charged, resulting in a false positive signal. It is likely necessary to remove particulate matter that could cause noise pollution.

[0069] The PNA beads are positioned very close to or directly below the glass chip 312 detector, and the film 304 It is pre-deposited on top. The glass chip 312 is gas without air bubbles being trapped on either side. The lath film 304 is attached in a moist state by any means that allows the film 3 It may be deposited on 04.

[0070] Here, we use probe-conjugate charged neutral polystyrene beads to detect specific nucleic acids. Please refer to Figure 4, which is Figure 400 of the detector of the device used for outputting. This is shown in Figures 3A to 3. This diagram was obtained from a photograph of device B in actual operation.

[0071] Exemplary procedure

[0072] Here, we use probe-conjugate charged neutral polystyrene beads to detect specific nucleic acids. Please refer to Figures 5A and 5B, which are flowcharts showing the method for obtaining the data.

[0073] In 502,

[0074] Cut the unbacked (double-sided water-permeable) Fusion5 film into strips of 2cm x 8cm. The fine particles generated during cutting are removed by gently blowing them with compressed air.

[0075] In 504,

[0076] A Pt foil electrode (0.7cm x 2cm) is placed on a glass microscope slide, and the electrode extends to the band. Place the Fusion5 film strip on top so that it is positioned directly below. The nail polish liquid is... It is used to join the end of an electrode with a wire to a slide and to seal around it. ru.

[0077] In 506,

[0078] The assembly consists of a neodymium magnet (approximately 1cm x 2cm x 0.2cm) with the magnet attached to it. It is positioned so that it is below the pole.

[0079] In 508,

[0080] Approximately 10 μL of magnetic PNA beads containing approximately 10 mg / mL is deposited on the film above the electrode. The stone should hold the beads in place.

[0081] In 510,

[0082] Place the filtered sample (approximately 200 μL or 4 drops) (in the sample loading area) The sample is added to one end of the Fusion5 film, and then the sample is followed along the film band onto the PNA beads. A sufficient buffer solution (10 mM NaCl, 25 mM Tris-HCl, pH 7) is needed for infusion. Add 0).

[0083] In 512,

[0084] Place a droplet of buffer solution onto the inverted glass tip. Then, quickly invert the tip and P Place the NA beads, magnet, and foil electrode directly on the Fusion5 film. Then, add one drop of buffer. The liquid is added to the reservoir on the top surface of the tip to cover the glass film, and the electrodes are placed in this reservoir. .

[0085] In 514,

[0086] After performing hybridization for about 1 minute, remove the magnet and apply approximately 1V to approximately 1V between the electrodes. A potential of 0.5V is applied. The baseline current is typically about 60nA to 100nA. .

[0087] In 516,

[0088] If the target NA is present in the sample, a decrease in the nA range of the current is expected within approximately 5 minutes. ru.

[0089] Further development

[0090] This method and the demonstration of the lateral flow apparatus were performed at a relatively low level of approximately 10,000 CFU / mL. This has only been done with high concentrations of E. coli samples. In future research, this setting... It may be possible to demonstrate a detection limit of at least 10 CFU / mL using this method. Therefore, the detection limit for rRNA is approximately 100 zM, which corresponds to approximately 1 CFU / 100 mL. Considering that this has only been demonstrated with the output device, it is still orders of magnitude greater than what might be possible. It's expensive.

[0091] Conceptually, a commercially available device similar to the one described above is envisioned. However, Fus The ion5 film band is likely to be incorporated dry within the disposable cartridge. , buffer salt pre-deposited in the sample pad area to control pH, and pre-deposited It is highly likely that it has magnetic or non-magnetic PNA beads. The magnet is incorporated into the base unit. It is highly likely that it is an embedded electromagnet. The absorption pad at the end opposite the sample pad is It is likely to be used to draw fluid through the Fusion5 membrane.

[0092] The interface between the glass chip and the Fusion5 film is a manufacturable cartridge It is unclear in the case of the cartridge. One method is after the cartridge has been inserted into the base unit. Until that point, keep the remaining part of the cartridge sealed and moist (no air bubbles) in the glass. The purpose is to accommodate the top.

[0093] Electronic devices maintain a potential of approximately 1V to 2V across the glass film while monitoring the current. A decrease in the current passing through the pores on the screen can be observed, which is a detected event. Double drops may be observed, which may be due to the clustering of multiple surrounding beads. .

[0094] While developing this system, cities like the one described in Koo's paper cited earlier... Nucleic acid was often extracted using commercially available kits. However, at a pH of approximately 10 for about 1 minute... It was also shown that sample dissolution, followed by filtration through approximately 0.1 μm and neutralization, appears to be appropriate. It is being developed (but has not yet been made public). Pre-loaded concentrated high pH buffer. Place approximately 1 mL of sample (urine, blood, saliva, sample) into a chamber containing (or dry buffer salt). A syringe-type sampling device has been developed that uses a cotton swab to draw up the buffer solution it has pressed into. After waiting for about 1 minute, push down the syringe, but only about 0. After passing through a 1 μm filter, a few drops of this dissolved and filtered sample enter the lateral flow zone. It deposits on top. In a preferred configuration, the dissolved sample flows over the PNA beads before p A dry neutralizing buffer is present on the band to neutralize H. Alternatively, neutralization is performed before filtration. Therefore, another chamber can be added to the syringe sampling device.

[0095] Further embodiments of lateral flow nucleic acid assays using an integrated pore-based detection system

[0096] 1. Introduction

[0097] This embodiment also relies on sustained pore blockage by a conjugated PNA capture probe. However, in this embodiment, it is not necessary for the pore-blocking polystyrene beads to be magnetic. do not have.

[0098] 2. Lateral flow band assembly

[0099] Now, refer to Figure 6, which is a side view of the lateral flow band assembly 600.

[0100] The assembly procedure steps are as follows:

[0101] A Cytiva A4-sized Fusion5 film sheet cut into 1.5cm x 3cm strips Cut it.

[0102] Use a paper cutter to cut the Cytiva backing card into 1.5cm x 8cm pieces. This will be the backing 602 for the assembly.

[0103] Cut Pt foil 604 into 2mm x 1cm strips, and solder wire 606 to the ends. Prepare the t-foil electrode 608.

[0104] Peel off the film from the backing card and solder it to the center of the backing 602 card. The prepared Pt foil electrode 608 is attached.

[0105] Two Fusion 5 film pieces are placed on the edges of the Pt foil electrode 608 and backing 602. The loading surface 610 and the absorption surface 612 are attached. These two Fusion5 film pieces A gap of less than 1 mm, 614, is left between them, exposing the Pt foil electrode downwards. The card is known as the lateral flow zone assembly 600.

[0106] PNA-modified beads are used in the lateral flow band assembly 600, as described in more detail below. It is positioned at point 616 on the loading surface 610.

[0107] One side of the chip is designated as the loading surface 610.

[0108] Hybridization of PNA / PEG / ethanolamine-modified polystyrene beads Buffer solution (10 mM NaCl, 25 mM Tris-HCl, pH 7, 1% Tween 2) Wash with 0).

[0109] The beads are concentrated by centrifugal filtration, and the beads are placed next to the gap 614 at the loading point 616. Load.

[0110] To hold the lateral flow membrane in place on the wall of the bath sonicator before the beads dry out Therefore, a vibrating force is applied to the beads. This step helps prevent bead aggregation.

[0111] Dry the beads.

[0112] 3. Glass Chip Assembly

[0113] Now, refer to Figures 7A to 7C. Figure 7A shows a side view of the glass chip assembly 700. This is a diagram. Figure 7B shows polydimethylsiloxane (PD) deposited on glass chip 704. MS) This is a diagram of the upper pattern 702. This glass chip 704 is thin, less than 1 μm thick. The region 706 is pre-etched to form a nanoscale region, which is then FIB processed. A pore size 708 is manufactured.

[0114] As shown in Figure 7A, the diameter must be smaller than the diameter of the beads used, approximately 1 μm. Glass chip 704, which has "nanopores" of approximately 800nm, has two PDMSO It is sandwiched in the center between the ring-shaped films 702 and 710. Cellophane tape (for example, S This tape is for easily removing dust from PDMS film 702 and 710. This is used to ensure good attachment to the glass chip 704.

[0115] Referring to Figure 7C, the polydimethylsiloxane deposited on the glass chip 704 A diagram of the bottom film 710 of the PDMS can be seen.

[0116] The bottom PDMS film 710 is approximately 0.3 mm thick and exposes nanopores 708. It has a circular opening 712 for inserting. The channel 714 extends from its edge to the circular opening 712. It is entirely formed of this material. This design allows air to escape when the underlying Fusion5 film gets wet. This allows for (see below) and tends to prevent the formation of bubbles. Upper PDMS Film 702 has a thickness of approximately 1 mm, and also has a circular opening 716 for exposing nanopores. It has. This circular opening 716 is a buffer reservoir for the upper electrode used for detection and It works by doing so (see below).

[0117] 4. The entire system assembly

[0118] Refer to Figures 6, 7A, and 8. Figure 8 shows the entire system assembly 800. This is a side view.

[0119] While using a Pt foil 604 electrode as the working electrode, both the counter electrode and the reference electrode are By using the Ag / AgCl electrode 804, the lateral flow band assembly Attach the 600 to the 802 potentiostat.

[0120] The glass chip assembly 700 is placed on top of the lateral flow band assembly 600. The pore 708 is positioned directly above the gap 614 within the lateral flow zone assembly 600.

[0121] A droplet of hybridization buffer 806 is placed on the glass tip assembly 700. Insert into the circular opening 716 of the MS upper pattern 702.

[0122] The above Ag / AgCl electrode 804 is placed on top of the above PDMS upper pattern 702. The buffer solution is gently lowered into the reservoir droplet 804 and into the glass tip assembly 700. An electrical connection is established through the nanopore 708.

[0123] 5. Detection

[0124] Approximately 400 μL of test sample 808 was loaded into the lateral flow zone assembly 600. The sample is deposited on surface 610. Due to the capillary flow, the liquid sample is directed to the target R in the sample. Point 616 so that NA or DNA hybridizes with the PNA probe on the modified bead. It flows over the PNA-modified beads that are arranged there.

[0125] The beads move more slowly than the fluid within the Fusion5 film, but at least some of them are gas It is transported to the gaps below the pores of the lath chip. The fluid passes through gap 614 to Fusion5 The fluid proceeds to the absorption surface 612 of the film. The fluid also flows through the lower PDMS O phosphorus beneath the glass tip 704. Load the opening of the shaped film 710, and ensure that no air bubbles form underneath. The PDMS O-ring shaped film 710 escapes through the channel 714.

[0126] It should be noted that this gap does not need to be completely free of any material. This allows the hybridized PNA beads to move sufficiently within the pore 114, blocking it. It must be open wide enough so that movement is not hindered (high porosity) (Sufficiently large pore diameter).

[0127] During this process, power is turned on to the potentiostat 802, and on the computer The data is collected using the following software.

[0128] Here, plot 9 shows the current in pore 708 observed by potentiostat 802. Refer to Figure 9, which is 00.

[0129] For reference, a "potentiostat" actually fixes the transformer pore voltage and current It could be a very simple device used to monitor the battery-powered voltage source and It can be as simple as a current monitor.

[0130] A stable baseline 902 current appears. In the case of a positive test, after a few minutes, nanopore 708 The sustained current is caused by PNA beads containing hybridized target nucleic acids that block the current. A decrease occurs, which is considered a detection signal. In a negative test, no decrease in current is observed. Only a stable baseline current exists.

[0131] Figure 9 shows the detection of 10 aM E. coli 16S rRNA in buffer solution. The following shows typical successful detection data achieved. The four exemplary detection signals are 904, 9 It is enclosed by 06, 908 and 910. The electric field polarity is determined by the first three detection signals 904, 9 It reversed after 06 and 908, and then returned after the first three events. Baseline 9 A return to 02 is observed, followed by the repetition signals 906, 908, and 910.

[0132] In this embodiment, it is not necessary to hold the magnetic PNA beads in place as in another embodiment. Therefore, it should be noted that magnet 322 in Figure 3A is no longer needed.

[0133] From the description herein, this disclosure includes, but is not limited to, several practical applications of the technology described herein. It will be understood that this encompasses the methods of implementation.

[0134] (i) a thin glass film and a glass chip having pores and (ii) a lateral flow film Bead-PN synthesis is achieved by controlling the embolization and bead position on the membrane, and by hybridizing the beads with the target nucleic acid. A magnetic beam is used to place the beads close to the glass chip for detection of the A conjugate. Use of PNA conjugates.

[0135] Lateral flow including a glass chip having an upper electrode, a lateral flow membrane, and a lower electrode. A low assay apparatus in which the glass tip is integrated with the lateral flow membrane. Ralphro assay apparatus.

[0136] A device for detecting specific nucleic acids, comprising: (a) a top surface, a bottom surface, a loading surface, and (b) a lateral flow membrane having an absorption surface, and (b) pores in contact with the upper surface of the lateral flow membrane. (c) A bottom electrode positioned at the bottom surface of the lateral flow membrane, and (d) positioned above the pores, (e) Wetting the lateral flow membrane on the loading surface, comprising an upper electrode immersed in a buffer solution. When a buffer solution is added to remove the pores, the lateral flow of the buffer solution cuts through the pores as it moves towards the absorption surface. (f) The buffer passes through and conducts a current that can be detected between the upper and lower electrodes. (g) When a voltage is applied between the upper and lower electrodes, the deposits are deposited in the pores, and when a voltage is applied between the upper and lower electrodes, the current passes through the pores. A device for detecting specific nucleic acids.

[0137] The pores are substantially circular, with a minimum diameter of approximately 500 nm and a typical height of less than approximately 1 μm. Apparatus of any preceding or succeeding embodiment, having a cylindrical to conical shape.

[0138] Apparatus of any preceding or subsequent embodiment, wherein the pores substantially contain borosilicate glass.

[0139] (a) Glass chip assembly, (i) typically having a thickness of about 1 μm or less (ii) the etched portion of the yacid glass and the portion located within the etched portion (iii) a pore and a circular opening whose center is above the pore and on the opposite side of the pore, The upper pattern of polydimethylsiloxane (PDMS) deposited on silicate glass, Apparatus of any preceding or subsequent embodiment further comprising a glass chip assembly including .

[0140] (a) One or more charged neutral peptide nuclei conjugated on polystyrene beads (b) The PNA capture probe further contains an acid (PNA) capture probe targeting pathogenic DNA / A device of any preceding or subsequent embodiment designed to capture RNA.

[0141] Any preceding or succeeding material whose pore diameter is smaller than the diameter of the polystyrene bead A device for implementation.

[0142] (a) further comprising a magnet adjacent to the deposition point of polystyrene beads, and (b) polystyrene beads The series contains magnetic material in part, and (c) the magnet attracts and holds the polystyrene beads. Apparatus of any preceding or subsequent embodiment.

[0143] A device for detecting specific nucleic acids, wherein (a) a lateral flow band assembly (1) backing, (2) loading surface placed on the backing, and (3) (4) The absorbing surface placed on the loading surface and the absorbing surface are electrically connected to both the loading surface and the absorbing surface. (5) The electrodes placed on the backing are in contact with the backing, and the electrodes placed between the loading surface and the absorption surface The gap is formed, and the gap is located above the electrode, and (6) the loading surface is filled. A lateral flow band assembly containing one or more peptide nucleic acid (PNA) beads. (b) a glass chip assembly, (1) a glass chip having a top surface and a bottom surface (2) an etched area less than 1 μm thick placed at the bottom of the glass chip (3) Nanopores arranged in the etched region of the glass chip, and (4) glass Polydimethylsiloxane (PDM) having a first circular opening located on the upper surface of the tip S) Top shape and (5) PD having a second circular opening located on the bottom surface of the glass chip MS bottom shape, including an open channel from a second circular opening to the edge of the shape, P (c) the entire system assembly, including the DMS bottom shape and the glass chip assembly. (1) A lateral flow band assembly attached to a glass chip assembly and (2) The gaps of the lateral flow assembly that align with the nanopores of the glass chip assembly. (3) Potato connected to the Ag / AgCl electrode positioned above the foil electrode and nanopores A system assembly including a stostat, and including a specific nucleic acid detection system The apparatus. Here, an Ag / AgCl electrode is used, but both the reference electrode and the counter electrode are... Alternative materials that can act simultaneously can be used.

[0144] (a) High (b) a droplet of bridization buffer, and (b) an Ag / AgC with one end immersed in the droplet. Apparatus of any preceding or subsequent embodiment further comprising an electrode and

[0145] A potentiostat measures the current passing through nanopores, any preceding or succeeding An apparatus for an embodiment of the above.

[0146] A device for detecting specific nucleic acids, comprising (a) a thin glass membrane and a glass having pores. (b) a lateral flow membrane in contact with the pores, and (c) magnetic beads-PNA (d) The position of the magnetic bead-PNA conjugate is such that the magnet Controlled on the membrane via (e) magnetic bead-PNA conjugate hybridizes For the detection of bead-PNA conjugates with target nucleic acids, the glass tip pores are positioned close to the bead. A device positioned to detect specific nucleic acids.

[0147] A method for detecting target nucleic acid (NA), wherein (a) unbacked Fu (b) Cutting the sion5 film into strips and removing the fine particles generated during cutting, and (b) foil electrodes Place the Fusi on a glass microscope slide so that the electrodes are positioned almost directly below the band. (c) The ON5 membrane is placed on top of the electrode, and the magnet is placed below the electrode. (d) Placing the lath slide on top of the neodymium magnet, and (d) a magnet on the film at a position above the electrode. The process involves depositing PNA beads, with the magnet holding the beads in place. (e) Add the dissolved and filtered sample to one end of the Fusion5 membrane, and Add enough buffer to push the sample down the membrane and onto the beads. (f) Placing a droplet of buffer solution on an inverted glass chip, and (g) turning the glass chip inverted Roll the glass chip onto the Fusion5 film directly above the beads, magnets, and foil electrodes. (h) Add a droplet of buffer solution to the reservoir on the top surface of the glass tip, and the upper electrode is connected to the reservoir. (i) Place the magnet on the bar and wait for hybridization to occur, then remove the magnet. (j) Applying a potential between electrodes, and if a target NA is present in the sample, a current A method for detecting target nucleic acid (NA), including the observation of a decrease in [a certain value].

[0148] A method for detecting a target nucleic acid (NA), comprising: (a) providing a lateral flow membrane including a top surface, a bottom surface, a loading surface , and an absorption surface; (b) providing pores in contact with the lateral flow membrane ; (c) providing a bottom electrode disposed on the bottom surface of the lateral flow membrane ; (d) providing a top electrode disposed above the pores, wherein the top electrode is immersed in a buffer solution; (e) dispensing the buffer solution to wet the lateral flow membrane of the loading surface , thereby allowing the lateral flow of the buffer solution to pass through the pores on the way to the absorption surface ; (f) the buffer solution is sufficiently deposited to conduct a detectable current between the top electrode and the bottom electrode; (g) when a voltage is applied between the top electrode and the bottom electrode <000090>, the current passes through the pores, a method for detecting a target nucleic acid (NA).

[0149] A method for detecting a target nucleic acid (NA), comprising: (a) providing a charge-neutral peptide nucleic acid (PNA) capture probe conjugated to polystyrene beads ; (b) providing pores in contact with the lateral flow membrane; (c) lysing the sample ; (d) filtering the lysed sample; (e) laterally flowing the lysed and filtered sample adjacent to the pores ; (f) applying a voltage across the pores ; (g) detecting an ionic current passing through the pores; (h) detecting a specific nucleic acid through a continuous decrease in the ionic current passing through the pores, a method for detecting a target nucleic acid (NA).

[0150] As used herein, the singular terms "a," "an," and "the" are used in context. Unless otherwise explicitly indicated, it can refer to multiple objects. Unless explicitly stated otherwise, the reference does not mean "one and the only one". Rather, it means "one or more."

[0151] The constructs of phrases such as "A, B and / or C" within this disclosure are not equivalent to A, B, or C. Describe whether any discrepancies exist, or any combination of items A, B, and C. A phrase construction that indicates "at least one" and then lists a group of elements is one such example. This indicates that at least one of the group elements exists, and that this is enumerated if applicable. Includes any possible combination of the elements.

[0152] The phrases "one embodiment," "at least one embodiment," or similar embodiments refer to this entire set of embodiments. References in the disclosure refer to specific features, structures, or other characteristics described in relation to the described embodiments. This indicates that the characteristics are included in at least one embodiment of the present disclosure. The phrases in the various embodiments do not necessarily all refer to the same embodiment or all other embodiments described. The phrase "embodiment" does not necessarily refer to a specific embodiment different from the given embodiment. A particular feature, structure, or characteristic of an embodiment may be one of the disclosed apparatus, system, or method. Or, it can be interpreted as meaning that they can be combined in any suitable way in multiple embodiments. It should be done.

[0153] As used herein, the term “set” refers to a collection of one or more objects. Therefore, for example, a set of objects can be a single object or multiple objects. It can include an object.

[0154] Related terms such as the first and second, top and bottom, up and down, left and right are not necessarily the same. Without requiring or implying any actual relationship or sequence between entities or actions, one It may be used solely to distinguish one entity or action from another entity or action.

[0155] "to include (comprises)", "to include (comprising)", "to "has", "having", "include es) '', including '', containing The terms "s)", "containing", or any of them Other variations are intended to cover non-exclusive inclusions, and as a result include lists of elements. Processes, methods, articles, or apparatus that include, possess, contain, or contain only those elements. This does not mean that, if not explicitly listed, such processes, methods, articles, or It may also include other elements specific to the device. "Includes a," "Has a," "Encloses a" The element following "contains" or "contains a" is, without further constraint, that includes that element. The presence of additional identical elements in a process, method, article, or apparatus that includes, contains, or comprises This does not exclude them.

[0156] As used herein, “approximately,” “approximately,” “substantially,” “essentially,” and The terms "approximately" or any other version thereof may be used to describe and explain minor variations. Used for the purpose of [doing something]. When used in conjunction with an event or situation, these terms refer to that event or situation. Refers to the case where an event or situation occurs exactly, as well as the case where the event or situation occurs approximately. It can be. When used in combination with a numerical value, the term can refer to a range of variation of ±10% or less of that numerical value, such as ±5% or less, ±4% or less, ±3 % or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.1% or less, or ±0.05% or less. For example, an aligned "Substantially" can refer to an angular variation within a range of ±10°, such as ±5°, ±4°, ±3°, ±2° or less, ±1°, ±0.5°, ±0.1°, or ±0.05° or less.

[0157] Furthermore, amounts, ratios, and other numerical values may be presented in range format in this specification. Such range format is understood to be used for convenience and brevity, and includes the explicitly specified numerical values as the limits of the range, but should be flexibly understood to also include all individual numerical values or sub - ranges subsumed within that range, whether or not each numerical value and sub - range is explicitly specified. For example, a ratio in the range of about 1 to about 200 includes the explicitly listed limits of about 1 and about 200, but should be understood to also include individual ratios such as about 2, about 3, and about 4, as well as sub - ranges such as about 10 to about 50 ​​​​​​​​​​​​​​​​​​​​​​​​​ Any element(s) that may make this more prominent are the technology or Any or all of the claims shall not be an important, necessary, or essential feature or element. It should not be interpreted that way.

[0160] Furthermore, in order to simplify this disclosure, various embodiments of the aforementioned disclosure are described in detail. The features can be grouped together. This disclosure method allows each claimed embodiment to be Reflecting the intention to require more features than those explicitly described in the claims It should not be interpreted as the subject matter of the present invention being more than the features of a single disclosed embodiment. This could also be a rare characteristic.

[0161] This summary of the disclosure is provided to enable readers to quickly confirm the nature of the technical disclosure. It shall not be used to interpret or limit the scope or meaning of the claims. It is submitted with an understanding of the content.

[0162] Implementation in certain jurisdictions may be limited to one or more parts of this disclosure after the filing of the application. It will be understood that the deletion of may be necessary. Therefore, readers should refer to the original content of this disclosure. Therefore, one should refer to the application filed at the time of filing. Any deletion of the contents of this disclosure shall not be made in the original application. It should not be interpreted as any abandonment, loss, or contribution to the public of any subject matter.

[0163] The following claims are incorporated herein by reference, and each claim is , it is an independent subject matter that can be claimed separately.

[0164] While this specification contains many details, these are not intended to limit the scope of this disclosure. It should not be interpreted, but merely provides examples of currently preferred embodiments. It should be interpreted as such. Therefore, the scope of this disclosure is limited to other information that may become apparent to those skilled in the art. It will be understood that this fully encompasses the embodiments.

[0165] All structural and functional equivalents to elements of disclosed embodiments known to those skilled in the art The object is expressly incorporated herein by reference and is included in the claims thereof. Intended. Furthermore, any element, component, or method step of this disclosure is an element, component, or The method step is public, whether or not it is explicitly stated in the claims. It is not intended to be made available to the public. The elements of the claims of this specification are Those elements are explicitly enumerated using the phrase "means for". Unless otherwise specified, "Means Plus Function" It should not be interpreted as an element of the claims of this specification. The element is explicitly stated using the phrase "step for". Unless otherwise listed, "Step Plus Function" It should not be interpreted as a "function)" element.

Claims

1. A device for detecting specific nucleic acids, (a) A lateral flow membrane having a top surface, a bottom surface, a loading surface, and an absorption surface, (b) Pores in contact with the upper surface of the lateral flow membrane, (c) A bottom electrode positioned on the bottom surface of the lateral flow membrane, (d) An upper electrode positioned above the pore and immersed in a buffer solution, Includes, (e) When a buffer solution is added to wet the lateral flow membrane on the loading surface The lateral flow of the buffer solution passes through the pores on its way to the absorption surface, (f) The buffer solution provides a current that can be detected between the upper electrode and the lower electrode. Sea urchins have accumulated sufficiently, (g) When a voltage is applied between the upper electrode and the lower electrode, the current flows through the pore Passing through, Device.

2. The pores are substantially cylindrical, having a minimum diameter of about 500 nm and a height of less than 1 μm. The apparatus according to claim 1, wherein the shape is conical.

3. The apparatus according to claim 2, wherein the pores substantially contain borosilicate glass.

4. (a) A glass chip assembly, (i) Etched portion of borosilicate glass with a thickness of approximately 1 μm or less, (ii) The pores located within the etched portion, (iii) A circular opening having its center above the pore and on the opposite side of the pore, Polydimethylsiloxane (PDMS) upper pattern deposited on borosilicate glass. and, The apparatus according to claim 3, further comprising a glass chip assembly including the following.

5. (a) One or more charged neutral peptide nuclei conjugated on polystyrene beads It further contains an acid (PNA) capture probe, (b) The PNA capture probe is designed to capture target pathogenic DNA / RNA. It is The apparatus according to claim 1.

6. The device according to claim 5, wherein the diameter of the pore is smaller than the diameter of the polystyrene bead. Place.

7. (a) further comprising a magnet adjacent to the deposition point of the polystyrene beads, (b) The polystyrene beads contain magnetite in part, (c) The magnet attracts and holds the polystyrene beads. The apparatus according to claim 6.

8. A device for detecting specific nucleic acids, (a) Lateral flow band assembly, (1) Backing and, (2) A loading surface placed on the backing, (3) an absorbent surface placed on the backing, (4) Electrically contacting both the loading surface and the absorption surface and on the backing The positioned electrodes, (5) A gap between the loading surface and the absorption surface, wherein the electrode The gap located above, (6) One or more peptide nucleic acids (PNAs) deposited at the position of the loading surface Beads and A lateral flow band assembly including, (b) A glass chip assembly, (1) A glass chip having an upper surface and a lower surface, (2) Etched region less than 1 μm thick located at the bottom of the glass chip and, (3) Nanopores arranged in the etched region of the glass chip, (4) Polydimethyl having a first circular opening disposed on the upper surface of the glass chip Siloxane (PDMS) top surface shape, (5) PDMS having a second circular opening located on the bottom surface of the glass chip The bottom shape includes an open channel from the second circular opening to the edge of the shape. , PDMS bottom shape and A glass chip assembly including, (c) The entire system assembly, (1) The lateral flow band assembly attached to the glass chip assembly Including Ri, (2) The gap of the lateral flow assembly is Aligned with the aforementioned nanopores, (3) Connected to the foil electrode and the Ag / AgCl electrode positioned above the nanopores Including a potentiostat, The entire system assembly, A device including a device.

9. (a) Distributed in the circular opening of the PDMS upper pattern of the glass chip assembly The placed droplets of hybridization buffer, (b) The Ag / AgCl electrode, with one end immersed in the droplet, An apparatus for detecting a specific nucleic acid according to claim 8, further comprising:

10. The potentiostat measures the current passing through the nanopores, as described in claim 9. A device for detecting specific nucleic acids.

11. A device for detecting specific nucleic acids, (a) A thin glass film and a glass chip having pores, (b) A lateral flow membrane in contact with the pores, (c) Magnetic beads-PNA conjugate and Includes, (d) The position of the magnetic bead-PNA conjugate is controlled on the film via a magnet. And so, (e) The magnetic bead-PNA conjugate hybridizes with the target nucleic acid. A glass chip positioned in close proximity to the pores for detecting the PNA conjugate. 、 Device.

12. Lateral flow including a glass chip having an upper electrode, a lateral flow membrane, and a lower electrode. A raw assay apparatus wherein the glass tip is integrated with the lateral flow membrane. A lateral flow assay device.

13. A method for detecting target nucleic acids (NAs), (a) Cut the unbacked Fusion 5 film into strips, and the microscopic particles generated during cutting Removing particles, (b) Place the foil electrode on a glass microscope slide and place the Fusion 5 film band on the electrode Furthermore, the electrodes are positioned so as to be located almost directly below the band, (c) The glass slide is placed on the neodymium magnet, and the magnet is placed below the electrode. To arrange them in such a way, (d) Depositing magnetic PNA beads on the film at a position above the electrode, The magnet should hold the beads in a predetermined position, (e) Add the dissolved and filtered sample to one end of the Fusion 5 membrane, and then the Add enough buffer to push the sample below the membrane and above the beads. That thing, (f) Placing droplets of buffer solution on an inverted glass chip, (g) Invert the glass chip and the Fus directly above the beads, magnet and foil electrode Placing the glass chip on the ion5 film, (h) Add a droplet of buffer solution to the reservoir on the upper surface of the glass chip, and the upper electrode is connected to the reservoir. To place it in the server, (i) After waiting for hybridization to occur, remove the magnet and between the electrodes Applying an electric potential, (j) When the target NA is present in the sample, a decrease in current is observed, Methods that include...

14. A method for detecting target nucleic acids (NAs), (a) To provide a lateral flow membrane including a top surface, a bottom surface, a loading surface, and an absorption surface. Toto, (b) To provide pores that come into contact with the lateral flow membrane, (c) To provide a bottom electrode disposed on the bottom surface of the lateral flow membrane, (d) To provide an upper electrode positioned above the pore, wherein the upper electrode is Being immersed in a buffer solution, (e) Dispense a buffer solution to wet the lateral flow membrane on the loading surface, As a result, the lateral flow of the buffer solution passes through the pores on its way to the absorption surface. To do so, (f) The buffer solution provides a current that can be detected between the upper electrode and the lower electrode. The fact that it accumulates sufficiently, (g) When a voltage is applied between the upper electrode and the lower electrode, the current flows through the pore To pass through, Methods that include...

15. A method for detecting target nucleic acids (NAs), (a) Charged neutral peptide nucleic acid (PNA) conjugated on polystyrene beads To provide a detection probe, (b) To provide pores that come into contact with the lateral flow membrane, (c) Dissolving the sample, (d) Filtering the dissolved sample, (e) Performing lateral flow of the dissolved and filtered sample adjacent to the pores Toto, (f) Applying a voltage across the pores, (g) Detecting the ion current passing through the pore, (h) Detecting specific nucleic acids through a sustained decrease in the ion current passing through the pores. Toto, Methods that include...

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