Integrated pore-based detection for lateral flow nucleic acid assay

Abstract

This device for lateral flow nucleic acid assays features an integrated pore-based detector, potentially enabling the detection of 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 ultralow concentrations (approximately 10 M to approximately 19 M). This scheme relies on the use of charge-neutral peptide nucleic acid (PNA) capture probes conjugated to polystyrene beads. The PNA beads acquire a substantial negative charge upon capture of the target pathogenic DNA / RNA, making them mobile in an electric field. Upon application of a bias voltage of approximately 1 V to 2 V, the PNA beads with hybridized targets are electrophoretically guided into a smaller-diameter pore. Subsequent pore blockage results in a strong and sustained decrease in the measured ionic current through the pore.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 075,669, filed on September 8, 2020, which is hereby incorporated by reference in its entirety.

[0002] [[ID=ll]]Description of Governmental Sponsorship Research or Development Not Applicable

[0003] Notice of Copyrighted Material Portions of the material in this patent document may be subject to copyright protection under the copyright laws of the United States and other countries. The copyright owner does not object to the reproduction by anyone of the patent document or patent disclosure, as it appears in the publicly available files or records of the U.S. Patent and Trademark Office, but reserves all other copyrights. The copyright holder does not waive any of its rights to keep this patent document confidential, including rights under 37 C.F.R.§1.14.

[0004] 1. Technical Field

[0005] The technology of the present disclosure generally relates to the detection of specific RNA or DNA fragments using complementary probes conjugated to charge - neutral polystyrene beads, and more particularly to the transverse flow detection of specific RNA or DNA fragments using complementary probes conjugated to charge - neutral polystyrene beads.

Background Art

[0006] 2. Background Explanation There is a strong push for the conception and development of low-cost, accurate, and robust point-of-care (POC) nucleic acid (NA)-based diagnostic devices that deliver results in minutes. Currently, most infectious disease diagnoses are achieved by culture methods, which typically take several days. While POC immunoassays are commercially available for pathogen detection, they often have limiting sensitivity and specificity, whereas less common nucleic acid (NA)-based tests have extremely low limit of detection (LOD), with both sensitivity and specificity ranging from 90% to 99%. A small number of POC NA-based tests are available for a limited number of analytes, including influenza, respiratory syncytial virus (RSV), and group A streptococcus.

[0007] NA-based testing for other indications is a clinical laboratory test, such as for Neisseria gonorrhoeae (NG, gonorrhea) and Chlamydia trachomatis (CT, chlamydia), and the process of transporting samples to the laboratory, batching, testing, and returning results typically takes several days. The key points regarding the significance of the technology disclosed here are summarized below.

[0008] Current methods for diagnosing most infectious diseases take more than a day, which can hinder the rapid administration of optimal treatment and counseling, lead to reliance on unreliable follow-up contact, result in inappropriate antibiotic prescriptions, cause prolonged patient suffering, and contribute to high healthcare costs.

[0009] Rapid determination of the presence or absence of important pathogens in clinical samples, i.e., qualitative testing, is of paramount importance, usually including influenza, RSV, SARS-CoV-2, HIV, human papillomavirus (HPV), NG, CT, etc.

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

[0011] The most important need is a qualitative test to determine the simple presence or absence (yes / no) of important pathogens in body fluids. Common pathogens that are abnormal in themselves at any level in body fluids and are indicators of infection include SARS-CoV-2, Group A Streptococcus, Neisseria gonorrhoeae, Chlamydia trachomatis, influenza virus, and Bordetella pertussis (pertussis).

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

[0013] Generally, optical methods are used for amplicon detection, which require the incorporation of optical components into the instrument due to increased complexity and cost. Despite remarkable advances in rapid methods for polymerase chain replication (PCR) cycling, the need for precise temperature control has led many test developers to pursue isothermal amplification methods. However, these methods still require extensive NA purification to remove primers, polymerase, and polymerase inhibitors, as well as reaction conditions that must be carefully controlled.

[0014] Widely applicable, NA-free, label-free, sequence-specific NA detection schemes are rare. In the last decade or so, there have been a few single-digit atomol (aM, 10⁻¹⁰) detection schemes. -18Significant progress has been made in developing novel approaches for amplified NA detection at clinically appropriate concentrations below the M range. However, only a handful of these schemes do not require special labeling other than oligonucleotides complementary to the target NA. Also, nearly half require some kind of optical system. The remaining approaches involve piezoelectricity, MALDI TOF MS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometry), or various electrochemical techniques.

[0015] Of these electrochemical detection-based schemes, only one uses simple and inexpensive potentiometer amperometry, but even that requires Pt nanoparticle labeling. Ideally, an NA sensor without amplification would contain only selective oligonucleotide probes, eliminating the need for further reagents, labeling, or complex signaling techniques. However, the latest technologies presented above suggest that detection schemes that meet this ideal are rare.

[0016] Previous research

[0017] The RNA / DNA detection device disclosed herein differs from other nanopore-based NA detection systems in that it is not a resistive pulse sensor based on the research of Coulter (DeBlois RW, Bean CP. Counting and Sizing of Submicron Particles by the Resistive Pulse Technique. Review of Scientific Instruments. 1970;41(7):909-16), where the conductance of electrolyte-filled pores or channels is monitored as various analytes pass through them. Rather, it is based on a much simpler conductivity measurement detection of large signals from long-lasting pore blockage.

[0018] While the resistance pulse technique focuses on the precise measurement of small changes in nanopore current over short timescales (μs~ms) as the analyte crosses the pore, the instrumental techniques disclosed herein essentially amplify this signal to the nA range and extend its duration indefinitely by relying on sustained pore blockade to signal the presence of the analyte.

[0019] This method greatly simplifies device electronics and readout, as described in PCT International Publication No. 2013 / 033647 by Monbouquette, Harold and Schmidt, Jacob, published on March 7, 2013, which is incorporated herein by reference in its entirety.

[0020] Nonspecifically bound NA yields little to no persistent signal. Note that a "signal" is a sustained, gradual decrease in ionic current lasting several seconds or longer. Most control executions by non-complementary NA do not result in observable pore blockade, with only a few transient blockades (not long enough to constitute a signal) being observed occasionally. However, incubation of beads with non-complementary NA has been observed to occasionally result in substantial nonspecific binding, as indicated by an increase in zeta potential from a single-digit range to approximately 20–30 mV. Therefore, these beads with nonspecifically bound DNA become negatively charged, electrophoretically mobile, and can be driven into pores.

[0021] At the pore opening, the electric field is strong enough to remove nonspecifically bound DNA from the beads, causing a decrease in bead charge and electrophoretic mobility. This allows the opposing force of electroosmotic flow to carry the beads out of the pore beyond the electrophoretic force. This electroosmotic flow arises from the counterflow of opposing ions against fixed negative charges on the glass pore wall.

[0022] Experimental evidence, as well as the results of many control studies, show that beads having only non-specifically bound NA approach the pore mouth for a short time and are then washed away by the opposing electroosmotic flow despite their potential mobility by dielectrophoresis. The literature does not appear to disclose another NA-based diagnostic system having such an active system to avoid false positives.

Prior Art Documents

Patent Documents

[0023]

Patent Document 1

Non-Patent Documents

[0024]

Non-Patent Document 1

Summary of the Invention

[0025] This technique describes a lateral flow nucleic acid assay using an integrated pore-based detector and methods of using the same.

[0026] In one embodiment, the technique described herein includes integrating a thin glass film and a glass chip having pores with a lateral flow film, and using a magnetic polystyrene bead-PNA (peptide nucleic acid) conjugate to control the position of the beads on the film and position the beads close to the glass chip to detect the bead-PNA conjugate by hybridized target nucleic acid. While magnetic polystyrene beads-PNA are used in this embodiment, it should be noted that other magnetic substrates that can be conjugated with charged neutral peptide nucleic acid (PNA) capture probes, including other charged neutral nucleic acid analogs, may also be used.

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

[0028] A key feature of the detector is the use of peptide nucleic acid (PNA) capture probes, which are uncharged polyamide analogs to NAs that share the same base chemistry. The bead-PNA conjugates are designed to be charge-neutral, so they do not exhibit electrophoretic motion sufficient to be detected in the presence of a DC electric field. However, the substantial negative charge acquired upon capture of the target NA sequence makes the hybridized conjugate mobile.

[0029] Electrophoresis of a bead-PNA conjugate with hybridized target NA into the mouth of a smaller diameter glass pore causes a significant increase in pore resistance, thereby resulting in a persistently strong, sustained decrease in the measured ion current. Nonspecifically bound NA is removed from the bead conjugate by the strong electric field within the pore mouth, resulting in no sustained signal. Furthermore, the opposite electroosmotic flow through the glass pore sweeps the PNA-bead conjugate away from the pore mouth without the hybridized target. In this way, this simple conductivity analyzer gives a highly selective (false-positive, rarely observed) binary response that signals the presence or absence of target NA (and associated pathogens).

[0030] Diagnostic applications of the apparatus and method include, but are not limited to, the following: 1. Any microorganism or viral pathogen, e.g., SARS-CoV-2, influenza, gonorrhea, chlamydia, RSV, Strep; 2. Use in clinics, emergency rooms, or emergency treatment centers; 3. COVID-19 screening, e.g., dental prostheses, surgical appointments, on-site, small conferences; 4. Home diagnostics; 5. Food safety; and 6. Foot-and-mouth disease (cattle).

[0031] Further military diagnostic applications may include, but are not limited to, diarrheal diseases; infectious wound assays; biological weapons agents, such as anthrax and plague; and site-specific pathogens, such as dengue fever or yellow fever.

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

[0033] Further aspects of the technology described herein are revealed in the following sections of this specification, and the detailed description is intended to fully disclose preferred embodiments of the technology without limitation.

[0034] The technology described herein will be better understood by referring to the following drawings. 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 is subjected to a positive voltage V + 104 and negative voltage V - It is positioned between 106 and 106.

[0037] This diagram shows polystyrene beads 108 having one or more covalently bonded peptide nucleic acid (PNA) probes 110 complementary to a single-stranded nucleic acid target (DNA or RNA, 112), and pores 114 passing through a glass membrane 102 with a diameter smaller than that of the beads 108. The beads 108 are purchased with carboxyl groups on their surface used as binding sites for the amine-terminated PNA probes 110. A particular bead 108 discussed in one embodiment has a diameter of 820 nm.

[0038] Other bead and pore dimensions, as well as geometric shapes, can function well. Submicron thick films performed best, but it was found that good performance is not limited to these dimensions when other materials are used. Furthermore, although cylindrical pores 114 were the target of the manufacturing process, substantially conical shapes were obtained. Other pore shapes can also function, as long as the minimum dimensions of the pores 114 are small enough that one or more hybridized beads can block the ionic current through the pores 114, resulting in a decrease in the pore current 114.

[0039] Initially, a specific single-stranded nucleic acid target (DNA or RNA, 112) is encapsulated in solution as an unbound portion. However, after some time, polystyrene beads 108 having one or more covalently bound peptide nucleic acid (PNA) probes 110 complementary to the specific single-stranded nucleic acid target (DNA or RNA, 112) achieve hybridization with those targets. This is shown at one or more locations on the bound polystyrene beads 116 where the covalently bound peptide nucleic acid (PNA) probes 118 are possibly bound to the single-stranded nucleic acid target (DNA or RNA, 112).

[0040] In the diagram in Figure 1, it can be seen that polystyrene beads 116 hybridized to three single-stranded nucleic acid targets (DNA or RNA, 112). This binding creates a network of many negative charges on the bound polystyrene beads 116 corresponding to the length of the target, thereby allowing the applied positive voltage V to penetrate. + 104 and negative voltage V - The applied electric field between 106 allows it to become electrophoretically mobile (or electrically mobile).

[0041] Typically, charged single-stranded nucleic acid targets (DNA or RNA, 112) can travel through the much larger pores 114 of the membrane 102 without interruption 122. Therefore, charged single-stranded nucleic acid targets (DNA or RNA, 122) can pass through the much larger pores 114 without visibly disturbing the ionic current passing through the pores 114.

[0042] In this diagram, the single-stranded nucleic acid target (DNA or RNA, 124) has already passed through the pore 114 of membrane 102.

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

[0044] However, RNA and DNA 112 have a considerable negative charge, and the complex has enough negative charge to move in an applied electric field, as shown in the example of the target RNA or DNA hybridizing to the PNA on the modified bead 120 and the bound polystyrene bead 116 (V - 106~V + 104).

[0045] As the PNA bead, which has a hybridized target resulting in a bound polystyrene bead 116, approaches the opening of the pore 114, a considerable and sustained deflection of the ion current occurs. This sustained decrease in the ion current is called "sustained."

[0046] Referring to Figures 2A to 2C, these are all prior art obtained from Koo B, Yorita AM, Schmidt JJ, Monbouquette HG. "Amplification-free, sequence-specific 16S rRNA detection at 1 aM." Lab Chip. 018;18(15):2291-9. doi:10.1039 / C8LC00452H.

[0047] Figure 2A is a photograph of a 1 cm square borosilicate glass sample with a microfabricated nanopore at the center of a thinly etched region.

[0048] Figure 2B is a scanning electron microscope (SEM) image of the nanopore-etched film shown in Figure 2A, viewed from an oblique angle.

[0049] Figure 2C is a SEM image of nanopores created by focused ion beam (FIB) in the etched film shown in Figure 2B. Such nanopores may be used as pore 114 in Figure 1.

[0050] Here, borosilicate glass has been used due to its wide applicability in the scientific community. However, the porous material can, in principle, be any material with a substantial fixed negative charge at a surface concentration so that it can generate electroosmotic flow to help prevent false positive test results. Furthermore, composite materials of two or more materials can also be used.

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

[0052] Figure 3A is a side view 300 of one embodiment of a lateral flow nucleic acid assay with integrated pore-based detection. A glass substrate, such as a borosilicate glass microscope slide, is used as the substrate 302. A membrane 304 having a bottom surface 306 and a top surface 308 is placed on the glass substrate 302. A sample loading region 310 is located on one side of the membrane 304.

[0053] The glass chip 312 contains micro or nanopores 114 manufactured as described above in Figure 1. These pores 114 are difficult to see in this figure because their diameter is approximately 500 nm. The glass chip 312 is attached to the film 304 on its upper surface 308 and conductively coupled to the platinum electrode 314 on its other surface using droplets of conductive buffer 316.

[0054] These glass tips 312 are incorporated into a disposable assay cartridge containing PNA beads and process fluid.

[0055] Between the film 304, the bottom surface 306, and the substrate 302, a platinum foil electrode 318 with a conductor 320 attached is also positioned. The platinum foil electrode 318 and the platinum electrode 314 are positioned to conduct a sensing current to the glass chip 312 when ions pass through the pores 114.

[0056] Although foil electrodes 318 are shown here, other electrode configurations can be used, such as simple wires, patterned wires, or even thin-film conductors directly deposited on the substrate 302.

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

[0058] An example of a membrane 304 is the Fusion5 membrane product from Cytiva (which is not lined on both sides to be water-permeable). Such a membrane 304 has an effective pore size large enough for either magnetic or non-magnetic PNA beads to move through it. With the Fusion5 membrane 304, a separate sample loading area 310 (made of a different material) is not required, but a separate sample pad can be used. If a larger liquid sample is used, an additional absorption pad can be added downstream of the glass detector to absorb excess liquid, thereby promoting flow along the lateral flow membrane.

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

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

[0061] Refer to Figures 3A, 3B, and 3C. The microfabricated nanopore glass chip 312 facilitates high-throughput manufacturing, a more direct interface to POC microfluidic devices, and the fabrication of low-cost devices. Such glass chips 312 were developed using MEMS (micro-electromechanical systems) processes to fabricate submicron-thick borosilicate glass films with pores 114 ranging from 100 nanometers to micron scales.

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

[0063] In another embodiment of a lateral flow nucleic acid assay having an integrated pore-based detector 300, a magnet 322 is used to maintain the position of polystyrene beads containing magnetite, thereby possessing ferromagnetic properties and being attracted to the magnet 322. It should be noted that other magnetic materials may be used to ferromagnetize the polystyrene beads. The magnet 322, which may be a neodymium magnet, another permanent magnet, or an electromagnet, is used to hold the magnetic PNA beads in place while the sample is drawn onto the beads for hybridization.

[0064] It should be noted that in Figure 3C, a top pattern 324 of polydimethylsiloxane (PDMS) is visible. This pattern is deposited on the upper surface of the glass chip 312 to better prevent droplets of conductive buffer 316 from spreading away from the platinum electrode 314. This is better achieved through the circular opening 326 located above the pores 114.

[0065] The target nucleic acid hybridizes to the PNA beads as the sample introduced into the sample loading region 310 flows over the PNA beads. The magnet 322 is then removed so that the beads with the hybridized target can move toward the glass tip and block the pores 114. Typically, the sample is moved by the capillary action of the membrane 304 upon addition of a chaser fluid that acts to "wash" the target toward the pores 114.

[0066] Consideration

[0067] This technology represents a potentially significant advance in NA detection, enabling low-cost, low-power (e.g., battery-powered), portable, compact, rapid, and robust devices. As part of the device, the detector is ideally integrated into the overall process flow for sample collection, cell lysis, NA extraction, and targeted NA hybridization to PNA probes on magnetic beads.

[0068] Sample collection and lysis are likely to be performed simultaneously and separately in syringes pre-loaded with lysis buffer. The sample (e.g., urine, blood) is drawn into the syringe, and lysis (i.e., chemical disruption of the microbial cell envelope or destruction of the viral capsid) occurs in approximately one minute. Subsequently, a few drops of the lysisd sample are deposited onto the sample pad area of ​​the assay device through a submicron filter (pore size approximately 0.1 μm) attached to the syringe. The filter is likely necessary to remove particulate matter that, if negatively charged, could cause pore blockage and lead to a false positive signal.

[0069] The PNA beads are pre-deposited on the film 304 at a position very close to or directly beneath the glass chip 312 detector. The glass chip 312 may be deposited on the film 304 by any means that allows the glass film 304 to be attached in a wet state without trapping air bubbles on either side.

[0070] Please refer to Figure 400, which is the detector of the apparatus for detecting specific nucleic acids using probe-conjugate charged neutral polystyrene beads. This is a diagram obtained from photographs of the apparatus in actual operation shown in Figures 3A and 3B.

[0071] Exemplary procedure

[0072] Please refer to Figures 5A and 5B, which are flowcharts 500 of a method for detecting specific nucleic acids using probe-conjugate charged neutral polystyrene beads.

[0073] In 502,

[0074] The unbacked (double-sided water-permeable) Fusion5 film is cut into 2cm x 8cm strips, and 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 Fusion5 film strip is placed on top so that the electrode is positioned almost directly beneath the strip. Nail polish is used to bond the end of the electrode, which has a solder wire, to the slide and to seal around it.

[0077] In 506,

[0078] The assembly is placed on top of a neodymium magnet (approximately 1cm x 2cm x 0.2cm) with the magnet positioned below the electrodes.

[0079] In 508,

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

[0081] In 510,

[0082] Add the filtered sample (approximately 200 μL or 4 drops) to one end of the Fusion5 membrane (within the sample loading area), and then add enough buffer (10 mM NaCl, 25 mM Tris-HCl, pH 7.0) to push the sample along the membrane onto the PNA beads.

[0083] In 512,

[0084] A droplet of buffer solution is placed on the inverted glass chip. The chip is then quickly inverted and placed on the Fusion5 film directly above the PNA beads, magnet, and foil electrode. Next, one drop of buffer solution is added to the reservoir on the top surface of the chip to cover the glass film, and the electrode is placed in this reservoir.

[0085] In 514,

[0086] After hybridization for approximately 1 minute, the magnet is removed and a potential of approximately 1V to 1.5V is applied between the electrodes. The baseline current is typically approximately 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.

[0089] Further development

[0090] This method and the lateral flow apparatus have only been demonstrated with relatively high E. coli (E. coli) sample concentrations of approximately 10,000 CFU / mL. Future studies may be able to demonstrate a detection limit of at least 10 CFU / mL using this setting, which is still orders of magnitude higher than what might be possible, given that a detection limit of approximately 100 zM of rRNA, equivalent to about 1 CFU / 100mL, has only been demonstrated with the detector alone.

[0091] Conceptually, a commercially available device similar to the one described above is envisioned. However, the Fusion5 membrane is likely to be dry-assembled within a disposable cartridge. The dry membrane is likely to have buffer salts pre-deposited in the sample pad area to control pH, as well as pre-deposited magnetic or non-magnetic PNA beads. The magnet is likely to be an electromagnet integrated into the base unit. An absorption pad at the end opposite the sample pad is likely to be used to draw fluid through the Fusion5 membrane.

[0092] The manner of the interface between the glass chip and the Fusion5 film is unclear in the manufacturable cartridge. One approach is to house the glass chip in a sealed, moist state (without air bubbles) away from the rest of the cartridge until a certain point after the cartridge has been inserted into the base unit.

[0093] The electronic device maintains a potential across the glass film at approximately 1V to 2V while monitoring the current. A decrease in the current passing through the pores on the screen can be observed, which is a detection event. Double droplets may be observed, which may be due to the clustering of multiple beads around the pores.

[0094] During the development of this system, nucleic acid extraction was often performed using commercially available kits, such as those described in Koo's paper cited earlier. However, it has also been shown that sample lysis at pH approximately 10 for about 1 minute, followed by filtration and neutralization through approximately 0.1 μm, appears to be appropriate (though this has not yet been published). A syringe-type sampling device has been developed in which approximately 1 mL of sample (urine, blood, saliva, or buffer containing a sampling swab) is drawn up into a chamber containing a pre-loaded concentrated high-pH buffer (or dry buffer salt). After waiting for about 1 minute, the syringe is pushed down, and the eluted lysed sample passes through an approximately 0.1 μm filter, with a few drops of this lysed and filtered sample accumulating on the lateral flow zone. In a preferred configuration, a dry neutralizing buffer is present on the zone to neutralize the pH before the lysed sample flows over the PNA beads. Alternatively, another chamber can be added to the syringe sampling device to perform neutralization before filtration.

[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, the pore-blocking polystyrene beads do not need to be magnetic.

[0098] 2. Lateral flow band assembly

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

[0100] The assembly procedure steps are as follows:

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

[0102] Using a paper cutter, cut the Cytiva backing card into 1.5cm x 8cm pieces. These will be backing 602 for assembly.

[0103] Pt foil 604 is cut into strips measuring 2 mm x 1 cm, and wire 606 is soldered to the ends to create Pt foil electrodes 608.

[0104] Remove the film from the backing card and attach the Pt foil electrode 608, which is soldered to the center of the backing card 602.

[0105] Two Fusion 5 film pieces, a loading surface 610 and an absorption surface 612, are mounted on the edges of the Pt foil electrode 608 and backing 602. The Pt foil electrode is exposed downwards, leaving a gap of less than 1 mm 614 between these two Fusion 5 film pieces. This assembled card is known as the lateral flow band assembly 600.

[0106] The PNA-modified beads are positioned at point 616 on the loading surface 610 of the lateral flow band assembly 600, as will be further detailed below.

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

[0108] Wash the PNA / PEG / ethanolamine-modified polystyrene beads with hybridization buffer (10 mM NaCl, 25 mM Tris-HCl, pH 7, 1% Tween 20).

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

[0110] Before the beads dry, a vibrating force is applied to them by holding the lateral flow membrane in place against the wall of the bath sonicator. This step helps prevent bead aggregation.

[0111] Dry the beads.

[0112] 3. Glass Chip Assembly

[0113] Refer to Figures 7A to 7C. Figure 7A is a side view of the glass chip assembly 700. Figure 7B shows the polydimethylsiloxane (PDMS) top pattern 702 deposited on the glass chip 704. This glass chip 704 is pre-etched to form a thinned region 706 less than 1 μm thick, and is subsequently FIB processed to produce nanopores 708.

[0114] As shown in Figure 7A, a glass chip 704 having "nanopores" 708 with a diameter of approximately 1 μm to 800 nm, which must be smaller than the diameter of the beads used, is sandwiched in the center between two PDMSO ring-shaped films 702 and 710. Cellophane tape (e.g., Scotch tape) is used to easily remove dust from the PDMS films 702 and 710, ensuring good adhesion to the glass chip 704.

[0115] Referring to Figure 7C, we can see a diagram of the polydimethylsiloxane (PDMS) bottom film 710 deposited on the glass chip 704.

[0116] The bottom PDMS film 710 is approximately 0.3 mm thick and has a circular opening 712 for exposing nanopores 708. The channel 714 is formed along its entire length from the edge to the circular opening 712. This design allows air to escape when the underlying Fusion5 film is wet (see below) and tends to prevent bubble formation. The top PDMS film 702 is approximately 1 mm thick and also has a circular opening 716 for exposing nanopores. This circular opening 716 acts as a buffer reservoir for the upper electrode used for detection (see below).

[0117] 4. The entire system assembly

[0118] Now, refer to Figures 6, 7A, and 8. Figure 8 is a side view of the entire system assembly 800.

[0119] The lateral flow band assembly 600 is attached to the potentiostat 802 by using the Pt foil electrode 604 as the working electrode and one of the Ag / AgCl electrodes 804 as both the counter electrode and the reference electrode.

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

[0121] A droplet 806 of hybridization buffer is placed into the circular opening 716 of the PDMS upper pattern 702 of the glass chip assembly 700.

[0122] The Ag / AgCl electrode 804 described above is gently lowered into the upper buffer reservoir droplet 804 within the PDMS upper pattern 702 described above, thereby establishing an electrical connection through the nanopores 708 within the glass tip assembly 700.

[0123] 5. Detection

[0124] Approximately 400 μL of test sample 808 is deposited onto the loading surface 610 of the lateral flow zone assembly 600. Due to the capillary flow, the liquid sample flows over the PNA-modified beads positioned at point 616 so that the target RNA or DNA in the sample hybridizes with the PNA probe on the modified beads.

[0125] The beads move through the Fusion5 film more slowly than the fluid, but at least some are carried into the gaps beneath the pores of the glass chip. The fluid then proceeds through the gaps 614 to the absorption surface 612 of the Fusion5 film. The fluid also loads into the openings of the lower PDMS O-ring shaped film 710 beneath the glass chip 704, and air escapes through the channels 714 of the lower PDMS O-ring shaped film 710 to prevent bubble formation.

[0126] It should be noted that this gap does not need to be completely free of any material. It must be sufficiently open (high porosity, sufficiently large pore diameter) so that the hybridized PNA beads can move freely within it and their movement to block pore 114 is not hindered.

[0127] During this process, the potentiostat 802 is powered on, and data is collected using software on a computer.

[0128] Here, refer to Figure 9, which is a plot of the current in pore 708 observed by potentiostat 802.

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

[0130] A stable baseline 902 current appears. In a positive test, after a few minutes, a sustained decrease in current occurs due to PNA beads containing hybridized target nucleic acids that block nanopores 708, and this is considered a detection signal. In a negative test, no decrease in current is observed, and only a stable baseline current is present.

[0131] Figure 9 shows typical successful detection data in which detection of 10 aM Escherichia coli (E. coli) 16S rRNA in buffer is achieved. Four exemplary detection signals are enclosed at 904, 906, 908, and 910. The field polarity reverses after the first three detection signals 904, 906, and 908, and then returns after the first three events. A return to baseline 902 is observed, followed by repeating signals 906, 908, and 910.

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

[0133] From the description herein, it will be understood that this disclosure encompasses multiple embodiments of the Technology, including but not limited to the following:

[0134] (i) Integration of a thin glass film and a glass chip having pores with (ii) a lateral flow film, and use of magnetic bead-PNA conjugates to control the position of beads on the film and position the beads close to the glass chip for detection of the bead-PNA conjugate with the hybridized target nucleic acid.

[0135] A lateral flow assay apparatus comprising a glass tip having an upper electrode, a lateral flow membrane, and a lower electrode, wherein the glass tip is integrated with the lateral flow membrane.

[0136] An apparatus for detecting a specific nucleic acid, comprising: (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 top surface of the lateral flow membrane; (c) a bottom electrode positioned on the bottom surface of the lateral flow membrane; and (d) an upper electrode positioned above the pores and immersed in a buffer solution, wherein (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 is deposited sufficiently to conduct a detectable current between the upper electrode and the bottom electrode; and (g) when a voltage is applied between the upper electrode and the bottom electrode, the current passes through the pores.

[0137] Apparatus of any preceding or subsequent embodiment, wherein the pores are substantially cylindrical to conical in shape, having a minimum diameter of about 500 nm and a typical height of less than about 1 μm.

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

[0139] (a) an apparatus of any preceding or subsequent embodiment further comprising a glass chip assembly comprising (i) an etched portion of borosilicate glass having a thickness of typically about 1 μm or less, (ii) pores located within the etched portion, and (iii) a top pattern of polydimethylsiloxane (PDMS) deposited on the borosilicate glass, including circular openings centered above and opposite the pores.

[0140] (a) an apparatus of any preceding or subsequent embodiment further comprising one or more charged neutral peptide nucleic acid (PNA) capture probes conjugated to polystyrene beads, and (b) the PNA capture probes are designed to capture target pathogenic DNA / RNA.

[0141] Apparatus of any preceding or subsequent embodiment, wherein the diameter of the pores is smaller than the diameter of the polystyrene beads.

[0142] (a) further comprising a magnet adjacent to the deposition site of polystyrene beads, (b) the polystyrene beads comprising a portion of magnetic material, and (c) the magnet attracting and holding the polystyrene beads, the apparatus of any preceding or subsequent embodiment.

[0143] A device for detecting specific nucleic acids, comprising: (a) a lateral flow band assembly comprising: (1) a backing; (2) a loading surface disposed on the backing; (3) an absorption surface disposed on the backing; (4) an electrode disposed on the backing in electrical contact with both the loading surface and the absorption surface; (5) a gap disposed between the loading surface and the absorption surface, the gap disposed above the electrode; and (6) one or more peptide nucleic acid (PNA) beads deposited at the position of the loading surface; and (b) a glass chip assembly comprising: (1) a glass chip having a top surface and a bottom surface; (2) an etched region less than approximately 1 μm thick disposed at the bottom of the glass chip; and (3) an etched region of the glass chip. A device for detecting a specific nucleic acid, comprising: (c) a glass chip assembly comprising: (4) a polydimethylsiloxane (PDMS) top shape having a first circular opening located on the top surface of the glass chip; and (5) a PDMS bottom shape having a second circular opening located on the bottom surface of the glass chip, including an open channel from the second circular opening to the edge of the shape; and (c) an entire system assembly comprising: (1) a lateral flow band assembly attached to the glass chip assembly; (2) gaps in the lateral flow assembly aligned with the nanopores of the glass chip assembly; and (3) a potentiostat connected to a foil electrode and an Ag / AgCl electrode located above the nanopores. Although an Ag / AgCl electrode is used here, alternative materials that can act simultaneously as both a reference electrode and a counter electrode can be used.

[0144] An apparatus of any preceding or subsequent embodiment further comprising: (a) a droplet of hybridization buffer placed in a circular opening of the PDMS upper pattern of a glass chip assembly; and (b) an Ag / AgCl electrode with one end immersed in the droplet.

[0145] A device of any preceding or succeeding embodiment in which a potentiostat measures current passing through nanopores.

[0146] An apparatus for detecting a specific nucleic acid, comprising (a) a glass chip having a thin glass membrane and pores, (b) a lateral flow membrane in contact with the pores, (c) a magnetic bead-PNA conjugate, (d) the position of the magnetic bead-PNA conjugate being controlled on the membrane via a magnet, and (e) the magnetic bead-PNA conjugate being positioned close to the glass chip pores for detection of the bead-PNA conjugate with a hybridized target nucleic acid.

[0147] A method for detecting a target nucleic acid (NA), comprising: (a) cutting an unbacked Fusion5 membrane into strips and removing microparticles generated during cutting; (b) placing a foil electrode on a glass microscope slide and positioning the Fusion5 membrane strip on the electrode so that the electrode is positioned approximately directly below the strip; (c) placing the glass slide on a neodymium magnet so that the magnet is positioned below the electrode; (d) depositing magnetic PNA beads on the membrane above the electrode, wherein the magnet should hold the beads in place; and (e) adding a dissolved and filtered sample to one end of the Fusion5 membrane. A method for detecting a target nucleic acid (NA), comprising: (f) adding enough buffer to push the sample below the membrane and onto the beads; (g) placing a droplet of buffer on an inverted glass tip; (h) inverting the glass tip and placing it on the Fusion5 membrane directly above the beads, magnet and foil electrode; (i) adding a droplet of buffer to the reservoir on the top surface of the glass tip and placing the upper electrode in the reservoir; (j) waiting for hybridization to occur, removing the magnet, and applying a potential between the electrodes; and (g) observing a decrease in current if the target NA is present in the sample.

[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 positioned on the bottom surface of the lateral flow membrane; (d) providing an upper electrode positioned above the pores, wherein the upper electrode is immersed in a buffer solution; (e) dispensing a buffer solution to wet the lateral flow membrane on the loading surface, thereby causing the lateral flow of the buffer solution to pass through the pores on its way toward the absorption surface; (f) depositing the buffer solution so as to conduct a detectable current between the upper electrode and the bottom electrode; and (g) when a voltage is applied between the upper electrode and the bottom electrode, the current passes through the pores.

[0149] A method for detecting a target nucleic acid (NA), comprising: (a) providing a charged neutral peptide nucleic acid (PNA) capture probe conjugated to a polystyrene bead; (b) providing a pore in contact with a lateral flow membrane; (c) dissolving a sample; (d) filtering the dissolved sample; (e) performing lateral flow of the dissolved and filtered sample adjacent to the pore; (f) applying a voltage across the pore; (g) detecting an ionic current passing through the pore; and (h) detecting a specific nucleic acid by a sustained decrease in the ionic current passing through the pore.

[0150] As used herein, the singular terms “a,” “an,” and “the” may refer to multiple objects unless explicitly indicated otherwise in the context. A singular reference to an object, unless explicitly stated, does not mean “one and only one,” but rather “one or more.”

[0151] The phrase constructs in this disclosure such as “A, B and / or C” indicate that A, B, or C may be present, or any combination of items A, B, and C. The phrase constructs indicating “at least one” followed by a list of elements indicate that at least one of these group elements is present, which, where applicable, includes any possible combination of the listed elements.

[0152] Any reference in this disclosure to “one embodiment,” “at least one embodiment,” or similar expressions of embodiment indicates that certain features, structures, or characteristics described in relation to the described embodiment are included in at least one embodiment of this disclosure. Therefore, these various embodiment phrases do not necessarily all refer to the same embodiment or a particular embodiment distinct from all other embodiments described. The embodiment phrase should be interpreted as meaning that certain features, structures, or characteristics of a given embodiment can be combined in any suitable way in one or more embodiments of the disclosed apparatus, system, or method.

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

[0154] Relational terms such as first and second, top and bottom, up and down, left and right may be used solely to distinguish one entity or action from another, without necessarily requiring or implying an actual relationship or order between such entities or actions.

[0155] The terms “comprises,” “comprising,” “has,” “having,” “includes,” “including,” “contains,” “containing,” or any other variation thereof are intended to encompass non-exclusive inclusion, and as a result, any process, method, article, or apparatus that includes, has, includes, or contains a list of elements does not include only those elements, but may also include other elements not expressly enumerated or that are specific to such process, method, article, or apparatus. The elements following “include a,” “have a,” “include a,” or “contain a” do not, without further restriction, exclude the existence of additional identical elements in any process, method, article, or apparatus that includes, has, includes, or contains that element.

[0156] Where used herein, the terms “approximately,” “approximately,” “substantially,” “essentially,” and “about,” or any other version thereof, are used to describe and explain small variations. Where used in conjunction with events or situations, these terms may refer to the event or situation occurring exactly, as well as the event or situation occurring approximately. Where used in conjunction with numerical values, the terms may refer to a range of variation of the numerical value of ±10%, such as ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%, or ±0.05%. For example, aligned “substantially” may refer to an angular variation of ±10°, such as ±5°, ±4°, ±3°, ±2°, ±1°, ±0.5°, ±0.1°, or ±0.05°.

[0157] Furthermore, quantities, ratios, and other numerical values ​​may be presented in range form as specified herein. Such range forms are understood to be used for convenience and brevity and include numerical values ​​explicitly designated as limits to the range, but should be understood flexibly to also include all individual numerical values ​​or subranges contained within that range, as if each numerical value and subrange were explicitly designated. 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 subranges such as about 10 to about 50, about 20 to about 100, etc.

[0158] As used herein, the term “combined” is defined as being connected, but not necessarily directly or mechanically. A device or structure “configured” in a particular way may be configured in at least that way, but in ways not enumerated.

[0159] No benefit, advantage, solution to a problem, or any element(s) that may cause or enhance any benefit, advantage, or solution is to be construed as an important, necessary, or essential feature or element of the technology described herein or any or all of the claims.

[0160] Furthermore, in the aforementioned disclosure, various features may be grouped together in various embodiments for the purpose of simplifying the disclosure. This method of disclosure should not be interpreted as reflecting an intention that the claimed embodiments require more features than are explicitly described in each claim. The subject matter of the present invention may consist of fewer features than all the features of a single disclosed embodiment.

[0161] This summary of the disclosure is provided to enable readers to quickly confirm the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the claims or their meaning.

[0162] It will be understood that implementation in certain jurisdictions may require the deletion of one or more parts of this disclosure after the filing of the application. Therefore, readers should refer to the application at the time of filing for the original content of this disclosure. No deletion of the content of this disclosure should be construed as an abandonment, loss, or contribution to the public of any subject matter of the original application.

[0163] The following claims are incorporated herein by reference and each claim is independent as separately claimed subject matter.

[0164] Although the description herein includes many details, these should not be construed as limiting the scope of this disclosure, but merely as providing examples of some currently preferred embodiments. Therefore, it will be understood that the scope of this disclosure fully encompasses other embodiments that may become apparent to those skilled in the art.

[0165] All structural and functional equivalents to elements of disclosed embodiments known to those skilled in the art are expressly incorporated herein by reference and are intended to be included within the claims. Furthermore, no elements, components, or method steps of this disclosure are intended to be made available to the public, whether expressly described in the claims or not. Elements of the claims herein should not be construed as "means plus function" elements unless they are expressly enumerated using the phrase "means for". Elements of the claims herein should not be construed as "step plus function" elements unless they are expressly enumerated using the phrase "step for".

Claims

1. A device for detecting specific nucleic acids, (a) A lateral flow membrane having a top surface, a bottom surface, a loading region, and an absorption edge, (b) A pore tip having a single pore, the pore tip 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 the sample solution is added to the lateral flow membrane in the loading region, the lateral flow of the sample solution passes through the pores on its way to the absorption edge, (f) The sample solution is sufficiently deposited to conduct a detectable current between the upper electrode and the lower electrode, (g) When a voltage is applied between the upper electrode and the bottom electrode, the current passes through the pore, (h) further comprising one or more peptide nucleic acid capture probes conjugated on polystyrene beads, (i) The peptide nucleic acid capture probe conjugated to a polystyrene bead is designed to capture target DNA or RNA from a sample solution deposited in the loading region of a lateral flow membrane, and the lateral flow causes the sample solution to flow over the peptide nucleic acid conjugated beads, and the target RNA or DNA in the sample solution hybridizes with the peptide nucleic acid probe on the beads. When peptide nucleic acid conjugate beads containing hybridized target DNA or RNA are subjected to electrophoresis at the entrance of the pores, the pore resistance increases significantly, thereby reducing the current detected between the upper and lower electrodes to a measurable level. The porous chip substantially comprises any material having a surface concentration of fixed negative charge sufficient to generate an electroosmotic flow capable of carrying away the bead-peptide nucleic acid conjugate without hybridized target DNA or RNA. Device.

2. The apparatus according to claim 1, wherein the pores are substantially cylindrical to conical in shape and have a length of less than 1 μm.

3. The pore chip is a glass chip assembly, (a) Including an etched portion of borosilicate glass with a thickness of less than 1 μm, (b) The pores are located within the etched portion, (c) The polydimethylsiloxane upper pattern is positioned on the tip and functions as a buffer reservoir for the upper electrode, and includes a circular opening in the center of the pores. (d) The apparatus according to claim 1, wherein the polydimethylsiloxane lower pattern is positioned below the tip and includes a circular opening in the center below the pores, having lateral channels for allowing air to escape from the opening to the end of the pattern.

4. The apparatus according to claim 1, wherein the diameter of the pore is smaller than the diameter of the polystyrene bead.

5. (a) further comprising a magnet or electromagnet adjacent to the deposition point of the polystyrene beads, (b) The polystyrene beads contain magnetite in part, (c) The magnet or electromagnet attracts and holds the polystyrene beads. The apparatus according to claim 4.

6. A method for detecting target RNA or DNA, (a) A lateral flow membrane including the top surface, bottom surface, loading region, and absorption edge, (b) A pore tip having a single pore, the pore tip in contact with 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, the upper electrode being immersed in the buffer solution, (e) A sample solution for wetting the lateral flow membrane arranged in the loading region, wherein the lateral flow of the sample solution passes through the pores on its way toward the absorption edge, Provided, (f) The sample solution is deposited sufficiently to conduct a detectable current between the upper electrode and the lower electrode, (g) When a voltage is applied between the upper electrode and the bottom electrode, the current passes through the pore, (h) further comprising one or more charged neutral peptide nucleic acid capture probes conjugated to polystyrene beads, (i) The peptide nucleic acid capture probe is designed to capture target DNA or RNA from a sample solution deposited in the loading region of a lateral flow membrane, and the lateral flow causes the sample solution to flow over the peptide nucleic acid conjugate beads, and the target RNA or DNA in the sample solution hybridizes with the peptide nucleic acid probe on the beads. When peptide nucleic acid conjugate beads containing hybridized target DNA or RNA are subjected to electrophoresis at the entrance of the pores, the pore resistance increases significantly, thereby reducing the current detected between the upper and lower electrodes to a measurable level. The porous chip substantially comprises any material having a surface concentration of fixed negative charge sufficient to generate an electroosmotic flow capable of carrying away the bead-peptide nucleic acid conjugate without hybridized target DNA or RNA. method.

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