Systems and methods for mobile device analysis of nucleic acids and proteins

Abstract

To provide a system for quick analysis of a biological sample.SOLUTION: The present invention discloses a portable system for extracting, optionally amplifying, and detecting nucleic acids or proteins using a compact integrated chip in combination with a mobile device system for analyzing detected signals, and comparing and distributing the results via a wireless network. Related systems and methods are also provided.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 61 / 951,084, filed Mar. 11, 2014; and claims the benefit of U.S. Provisional Patent Application No. 61 / 875,661, filed Sep. 9, 2013; and claims the benefit of U.S. Provisional Patent Application No. 61 / 790,354, filed Mar. 15, 2013. The entire teachings of the above provisional patent applications are incorporated herein by reference.

Background Art

[0002] The detection and analysis of genetic material of biological organisms can be used for pathogen identification, genotyping, biomarker identification, personalized medicine, personalized nutrition, personalized skin care, personalized pharmaceutical cosmetics, personalized nutritional supplements, companion diagnostics, drug monitoring, pharmacogenetics, and nutrigenomics. The identity of a species in a biological sample can be confirmed by comparing the nucleic acid present in the sample with the nucleic acid of a known reference sample. However, before performing this comparison, the nucleic acid must be extracted, amplified, and then detected from the sample. Typically, the extraction, amplification, and detection steps are performed in a laboratory or hospital over a period of hours, days, or weeks. For example, amplification is typically related to the polymerase chain reaction (PCR) as described in U.S. Pat. Nos. 4,683,202 and 4,683,195. To amplify nucleic acid using conventional PCR, the nucleic acid must be repeatedly heated and cooled in the presence of enzymes, nucleotides, primers, and buffers.

[0003] Conventional methods and devices for nucleic acid extraction, amplification, and detection are generally not robust enough to be used in mobile or field environments outside of specialized laboratory facilities. Extraction and amplification alone can take several hours, if not several days, depending on the type of organism, nucleic acid chain length, and number of cycles. Furthermore, commercially available devices and methods require skill, running water, and electricity. Additionally, temperature, pH, and buffer components must be carefully controlled. Contaminants can inhibit or interfere with the nucleic acid polymerase enzyme used in replication, reducing the efficiency and fidelity of the amplification process. Similar limitations apply to conventional techniques for nucleic acid extraction and detection. Therefore, there is a need for automated, integrated, compact, robust, rapid, accurate, and user-friendly devices and methods for detecting, quantifying, and identifying nucleic acids. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent No. 4,683,202 [Patent Document 2] U.S. Patent No. 4,683,195 [Overview of the project]

[0005] The present invention relates to a system and method for the rapid analysis, quantification, and identification of nucleic acids or proteins.

[0006] The present invention provides a portable, mobile, or point-of-care (POC) system for extracting, optionally amplifying, and detecting (and optionally quantifying) nucleic acids or proteins using a compact integrated chip in conjunction with a portable system or mobile device for detection signal analysis, and for comparing and supplying the results via a wireless network. Interchangeable modules may be used for one or more of the extraction, amplification, and detection functions.

[0007] The present invention offers several advantages. It is faster, cheaper, field-deployable, and more accurate than existing methods for detecting and analyzing nucleic acids, or in some embodiments, proteins. Unlike conventional methods that can take hours or weeks to characterize or analyze even large samples, the present invention typically produces measurements within an hour, 30 minutes, or even minutes. The present invention is sufficiently sensitive to detect nucleic acids extracted from samples containing a small number of cells (see examples). Unlike standard diagnostic devices and methods that require benchtop equipment in a laboratory or clinic, skilled technicians, electricity, water, and often refrigeration of samples and reagents, the present invention can be implemented in portable or mobile devices using compact, disposable integrated chips. Results can be delivered via one or more information transmission networks, such as wireless networks and / or the Internet. The system of the present invention does not require skilled or proficient technicians and can function outside of hospitals or centralized laboratory facilities. The system of the present invention is robust to a variety of environmental variables and can function over a wide range of pH, temperature, conventional overhead equipment, and optionally without refrigeration.

[0008] The present invention can be used to detect and distinguish nucleic acid molecules or proteins from a single biological organism or other source, or from multiple organisms or sources. Not only can the present invention detect and analyze unknown nucleic acids, but the method is also sufficiently sensitive to distinguish species within a genus or to distinguish point mutations and / or biomarker sequences and / or disease susceptibility alleles. Embodiments of the present invention using disposable integrated chips also offer significant cost savings compared to existing diagnostic assays.

[0009] Furthermore, in embodiments of the present invention using a non-thermal nucleic acid amplification process as disclosed in U.S. Patent No. 7,494,791 (the entire disclosure is incorporated herein by reference), approximately 1 × 10⁶ per base pair. -7 Less than or better than the error (e.g., 10 per base pair) -10An error rate of less than 10⁻¹⁵ can be achieved. Even with "difficult sequences" (sequences that tend to cause polymerase enzymes to shift, produce errors, or stop functioning; examples of difficult sequences include repetitive sequences, poly-A sequences, GC-rich sequences, and trinucleotide repetitive sequences), an error rate of 1 × 10⁻¹⁵ per base pair can be achieved. -3 Less than or better than the error (e.g., 10 per base pair) -6 Error rates of less than 100 can be achieved. Using reagents capable of surviving more than 100 nucleic acid replication cycles, sequences of up to 20,000 base pairs in length can be amplified using the disclosed non-thermal nucleic acid amplification method. Other isothermal amplification methods may be used, such as Loop-Mediated Isothermal Amplification (LAMP) techniques, as described in T. Notomi, et al., Nucleic Acids Research, 28, e63 (2000); Helicase-Dependent Amplification (HDA) techniques, as described in Vincent M, Xu Y, Kong H. (2004), "Helicase-dependent isothermal DNA amplification," EMBO Rep 5 (8): 795-800; and Strand Displacement Amplification (SDA) techniques, as described in GT Walker, et al., Proc. Natl. Acad. Sci USA, 89, 392-396 (1992); all of these teachings are incorporated herein by reference in their entirety. Bridge, rolling circle, and any other amplification method (thermal or isothermal) may be used.

[0010] Embodiments according to the present invention provide a system for the rapid analysis of biological samples. The system comprises a mobile device that receives at least one integrated chip. The mobile device processes the integrated chip and analyzes a biological sample loaded thereon. Together, the mobile device and the integrated chip are configured to perform at least one of the manipulation and control of molecules or fluid systems on the integrated chip. Together, the mobile device and the integrated chip are configured to precisely control at least one parameter that governs at least one of a plurality of steps in the analysis of a biological sample to within plus or minus 10%, plus or minus 1%, plus or minus 0.1%, plus or minus 0.01%, plus or minus 0.001%, or plus or minus 0.0001%.

[0011] In another embodiment according to the present invention, a system for rapid analysis of biological samples is provided. The system comprises a portable control assembly that receives at least one compact integrated chip. The integrated chip comprises an extraction module; which may comprise a nucleic acid amplification module fluidly connected to the extraction module; and a biological sample detection module fluidly connected to either the nucleic acid amplification module or the extraction module. The portable control assembly processes the integrated chip and analyzes a biological sample loaded thereon by using: the extraction control module; the nucleic acid amplification control module functionally connected to the extraction control module; and the biological sample detection control module functionally connected to both the nucleic acid amplification module and the extraction module.

[0012] Another embodiment according to the present invention provides a system for rapid analysis of biological samples. The system comprises a portable control assembly that receives at least one compact integrated chip. The integrated chip comprises an injection port for loading a biological sample and optionally one or more reagents onto the integrated chip; an extraction module; a nucleic acid amplification module fluidly connected to the extraction module; and a detection module fluidly connected to the nucleic acid amplification module. The portable assembly comprises an extraction control module, which in some embodiments comprises, for example, magnetic particle capture means for capturing magnetic particles introduced into the biological sample loaded onto the integrated chip; a nucleic acid amplification control module functionally connected to the extraction control module, wherein the nucleic acid amplification means in some embodiments comprises, for example, thermoelectric, thin-film, infrared, or optical-based or mechanical heating means for heating / cooling the nucleic acid amplification module of the integrated chip; and a detection control module functionally connected to the nucleic acid amplification module and the nucleic acid extraction module. In some embodiments, the detection control module comprises fluorescence detection means for detecting nucleic acids and proteins; and in some embodiments, capillary electrophoresis (CE) control means functionally coupled to the fluorescence detection means, further comprising a high-voltage control unit for applying a voltage sufficient to perform the separation of nucleic acids or proteins over the entire area of ​​the nucleic acid detection module on the integrated chip; and fluid pressure generating means for moving a biological sample and / or nucleic acids through the integrated chip.

[0013] Another embodiment according to the present invention provides a method for rapid analysis of a biological sample. The method includes: (1) preparing at least one integrated chip comprising a nucleic acid extraction module; a nucleic acid amplification module fluidly connected to the nucleic acid extraction module; and a nucleic acid detection module fluidly connected to the nucleic acid amplification module; (2) loading at least one biological sample onto at least one integrated chip; (3) functionally connecting at least one portable control assembly comprising a nucleic acid extraction control module; a nucleic acid amplification control module functionally connected to the nucleic acid extraction control module; and a nucleic acid detection control module functionally connected to the nucleic acid amplification module and the nucleic acid extraction module; and (4) operating the portable control assembly to perform extraction, amplification, and detection of nucleic acids from the biological sample loaded onto the integrated chip.

[0014] Another embodiment according to the present invention provides a method for rapid analysis of a biological sample. The method includes: (1) preparing at least one integrated chip comprising a protein extraction module and a protein detection module fluidly connected to the protein extraction module; (2) loading at least one biological sample onto at least one integrated chip; (3) functionally connecting at least one portable control assembly comprising a protein extraction control module and a protein detection control module functionally connected to the protein extraction module to at least one integrated chip; and (4) activating the portable control assembly to perform the extraction and detection of proteins from the biological sample loaded onto the integrated chip.

[0015] In another embodiment according to the present invention, an integrated chip is provided for rapid sequential extraction, amplification, and separation of nucleic acids from a biological sample. The integrated chip comprises a housing integrating therein microfluidic channels continuously fluid-communicated to a nucleus extraction module, a nucleic acid amplification module, and a nucleic acid separation module; and a sample inlet port fluid-communicated to the nucleic acid extraction module for injecting a biological sample and reagents; wherein the nucleic acid extraction module comprises at least one extraction chamber for extracting nucleic acids from a biological sample, the extraction chamber being connected to the sample inlet port by at least one sample transport channel; wherein the nucleic acid amplification module comprises at least one amplification chamber for amplifying nucleic acids, the nucleic acid amplification chamber being connected to the extraction chamber by at least one nucleic acid transport channel; and wherein the nucleic acid separation module comprises at least one detection channel for separating and detecting nucleic acids, the detection channel being connected to the nucleic acid amplification chamber by at least one amplification product transport channel.

[0016] Another embodiment according to the present invention provides a method for rapid analysis of a biological sample. The method includes preparing at least one integrated chip, the integrated chip comprising: a nucleic acid extraction module; a nucleic acid amplification module fluidly connected to the nucleic acid extraction module; and a nucleic acid detection module fluidly connected to the nucleic acid amplification module. The method further includes loading at least one biological sample onto at least one integrated chip; and functionally connecting a portable control assembly to at least one integrated chip. The portable control assembly comprises a nucleic acid extraction control module; a nucleic acid amplification control module functionally connected to the nucleic acid extraction control module; and a nucleic acid detection control module functionally connected to the nucleic acid amplification module and the nucleic acid extraction module. The method further includes activating the portable control assembly to perform extraction, amplification, and detection of nucleic acids from the biological sample loaded onto the integrated chip; and determining at least one biomarker related to a human being, which is the source of at least one biological sample, based on the detection of nucleic acids from the biological sample.

[0017] In further relevant embodiments, the method may include determining at least one of the following based on at least one biomarker: (i) a dosage of at least one drug to perform a treatment for a disease condition associated with at least one biomarker; (ii) a combination of several drugs to perform a treatment for a disease condition associated with at least one biomarker; and (iii) a determination of whether a person who is the source of at least one biological sample will respond to drug treatment for a disease condition associated with at least one biomarker.

[0018] In other related embodiments, the method may include determining the amount of at least one biomarker associated with the person from whom the at least one biological sample is derived, based on the detection of nucleic acids from the biological sample; and determining the degree of treatment progression of a disease condition associated with the at least one biomarker in the person from whom the at least one biological sample is derived, based on the amount of at least one biomarker in the at least one biological sample.

[0019] In further relevant embodiments, the method may include determining at least one biomarker associated with a person who is the source of at least one biological sample, based on the detection of nucleic acids from the biological sample; and determining, based on the at least one biomarker, at least one of the following: (i) selection of personal care products for the person who is the source of at least one biological sample; (ii) the amount of personal care products to be delivered for that person; (iii) the cosmetic skin type of that person, for example; and (iv) the amount of at least one cosmetic biomarker of that person in at least one biological sample.

[0020] In other related embodiments, the method may include determining the amount of at least one cosmetic biomarker associated with the person from whom the at least one biological sample is derived, based on the detection of nucleic acids from the biological sample; and determining the degree of progression of a cosmetic procedure in the person from whom the at least one biological sample is derived, based on the amount of at least one biomarker in the at least one biological sample.

[0021] In further relevant embodiments, the method may include determining at least one biomarker associated with a person who is the source of at least one biological sample, based on the detection of nucleic acids from the biological sample; and determining the degree or type of health of the person who is the source of at least one biological sample, based on the at least one biomarker.

[0022] In other relevant embodiments, the method may include determining at least one biomarker associated with a person who is the source of at least one biological sample, based on the detection of nucleic acids from the biological sample; and determining, based on the at least one biomarker, whether the person who is the source of at least one biological sample is a member of a subset of genes with respect to at least one biomarker.

[0023] In further relevant embodiments, the method may include determining at least one biomarker associated with a person who is the source of at least one biological sample, based on the detection of nucleic acids from the biological sample; and determining at least a portion of a personalized genomic profile of the person who is the source of at least one biological sample, based on the at least one biomarker.

[0024] In further relevant embodiments, determining at least a portion of a personalized genomic profile may include detecting nucleic acids relating to a person's wellness, their sports nutrition, their personalized diet, and their nutrition or personalized nutrition.

[0025] In another embodiment according to the present invention, a system for rapid analysis of a biological sample is provided. The system comprises at least one integrated chip, the integrated chip comprising: a nucleic acid extraction module; a nucleic acid amplification module in fluid communication with the nucleic acid extraction module; and a nucleic acid detection module in fluid communication with the nucleic acid amplification module, and at least one biological sample is loaded onto the at least one integrated chip. The system further comprises a portable control assembly comprising: a nucleic acid extraction control module; a nucleic acid amplification control module functionally linked to the nucleic acid extraction control module; and a nucleic acid detection control module functionally linked to the nucleic acid amplification module and the nucleic acid extraction module, the portable control assembly performing extraction, amplification and detection of nucleic acids from the biological sample loaded on the integrated chip. The system further comprises a genetic analysis unit configured to determine at least one biomarker associated with a person who is the source of the at least one biological sample based on the detection of nucleic acids from the biological sample. The genetic analysis unit is further configured to determine at least a portion of an individualized genomic profile of a person who is the source of the at least one biological sample based on the at least one biomarker.

[0026] In another embodiment according to the present invention, a system for rapid analysis of a biological sample is provided. The system comprises at least one integrated chip, the integrated chip comprising: a nucleic acid extraction module; a nucleic acid amplification module in fluid communication with the nucleic acid extraction module; and a nucleic acid detection module in fluid communication with the nucleic acid amplification module, with at least one biological sample being loaded onto the at least one integrated chip. The system further comprises a portable control assembly comprising: a nucleic acid extraction control module; a nucleic acid amplification control module functionally linked to the nucleic acid extraction control module; and a nucleic acid detection control module functionally linked to the nucleic acid amplification module and the nucleic acid extraction module, the portable control assembly performing extraction, amplification and detection of nucleic acids from the biological sample loaded on the integrated chip. The system further comprises a genetic analysis unit configured to determine at least one biomarker associated with a person who is the source of the at least one biological sample based on detection of nucleic acids from the biological sample; and a heartbeat control unit linked to the genetic analysis unit and configured to control enhanced external counterpulsation (EECP) or other heartbeat parts based on the at least one biomarker.

[0027] Embodiments may perform protein separation, such as by electrophoresis.

[0028] Further related systems and methods are provided.

[0029] As will be apparent from a more detailed description of exemplary embodiments of the invention described below, as shown in the accompanying drawings in which like reference numerals refer to the same parts throughout various views, the drawings are not necessarily to scale, but rather place emphasis on illustrating embodiments of the invention.

Brief Description of the Drawings

[0030] [Figure 1] FIG. 1 illustrates an embodiment of a portable assay or mobile system having a compact integrated chip for detecting and analyzing nucleic acids. [Figure 1A] Figure 1A illustrates an example of a modular design for a portable assay system that communicates with a genome database. [Figure 2] Figure 2 illustrates an embodiment of the modular design of the system and device of the present invention. [Figure 2A] Figure 2A shows an embodiment of the module design of the extraction control module. [Figure 2B] Figure 2B shows an embodiment of the module design of the amplification control module. [Figure 3] Figure 3 is an illustrative example of a flowchart for a method of detecting and analyzing nucleic acids. [Figure 4] Figure 4 illustrates an embodiment of a compact integrated chip that may be used in conjunction with the device and method of the present invention. [Figure 5] Figure 5 shows an exemplary passive plug that controls fluid flow on an integrated chip. [Figure 6A] Figure 6A shows an exemplary electromagnetically controllable valve and a method for controlling the flow of fluid through an integrated chip used in the present invention. [Figure 6B] Figure 6B shows an exemplary electromagnetically controllable valve and a method for controlling the flow of fluid through an integrated chip used in the present invention. [Figure 7A] Figure 7A shows an exemplary method for controlling the flow of fluid in and out of an on-tip well. [Figure 7B] Figure 7B shows an exemplary method for controlling the flow of fluid in and out of an on-tip well. [Figure 8A] Figure 8A shows an exemplary arrangement of channels and a method for injecting a sample into the channels used for capillary electrophoresis according to embodiments of the present invention. [Figure 8B] Figure 8B shows an exemplary arrangement of channels and a method for injecting a sample into the channels used for capillary electrophoresis according to embodiments of the present invention. [Figure 8C] Figure 8C shows an exemplary arrangement of channels and a method for injecting a sample into the channels used for capillary electrophoresis according to embodiments of the present invention. [Figure 9A] Figure 9A shows an embodiment of the module design of the fluorescence detection control module. [Figure 9B] Figure 9B shows an exemplary system and method for detecting a fluorescence signal generated by a nucleic acid detection module on an integrated chip. [Figure 10] Figure 10 is a flowchart illustrating the applicability of the present invention in various applications. [Figure 11] Figure 11 shows a compact integrated chip and a hardware module for use according to an embodiment of the present invention. [Figure 12] Figure 12 shows the hardware module from Figure 11 and the Peltier heating device for use with it. [Figure 13] Figure 13 shows a compact integrated chip and a detection system for use with it. [Figure 14] Figure 14 shows a compact integrated chip and a hardware system for use according to an embodiment of the present invention. [Figure 15A] Figure 15A shows a different view of a microfluidic valve in a compact integrated chip based on an expandable, encapsulating elastic membrane. [Figure 15B] Figure 15B shows a different view of a microfluidic valve in a compact integrated chip based on an expandable, encapsulating elastic membrane. [Figure 16A] Figure 16A shows a different view of a microfluidic valve assembly in a compact integrated chip based on an expandable, encapsulating elastic membrane. [Figure 16B] Figure 16B shows a different view of a microfluidic valve assembly using an expandable, encapsulating elastic membrane-based compact integrated chip. [Figure 16C] Figure 16C shows a different view of a microfluidic valve assembly using an expandable, encapsulating elastic membrane-based compact integrated chip. [Figure 16D] Figure 16D shows a different view of a microfluidic valve assembly on a compact integrated chip based on an expandable, encapsulating elastic membrane. [Figure 16E] Figure 16E shows a different view of a microfluidic valve assembly using an expandable, encapsulating elastic membrane-based compact integrated chip. [Figure 16F] Figure 16F shows a different view of a microfluidic valve assembly using an expandable, encapsulating elastic membrane-based compact integrated chip. [Figure 17A] Figure 17A shows the results of on-chip DNA purification and amplification. [Figure 17B] Figure 17B shows the results of on-chip DNA purification and amplification. [Figure 17C] Figure 17C shows the results of on-chip DNA purification and amplification. [Figure 17D] Figure 17D shows the results of on-chip DNA purification and amplification. [Figure 18] Figure 18 shows a depiction of agarose gel-based electrophoresis confirming the size of the resulting amplification product. [Figure 19A] Figure 19A shows the results of on-chip DNA purification and amplification. [Figure 19B] Figure 19B shows the results of on-chip DNA purification and amplification. [Figure 19C] Figure 19C shows the results of on-chip DNA purification and amplification. [Figure 19D] Figure 19D shows the results of on-chip DNA purification and amplification. [Figure 20] Figure 20 shows a depiction of agarose gel-based electrophoresis confirming the size of the resulting amplification product. [Figure 21] Figure 21 shows a wearable device. (A) is a skin patch or dermal patch; (B) is a bracelet comprising the device of the present invention and a band for holding the device on the wrist, arm, foot, or fingers; and (C) is a patch on which one side is adhesive and attachable to the outside of the body, and the same or opposite side is adhesive and attachable to the device of the present invention. [Figure 22] Figure 22 shows a device configured as an implantable device, injectable device, or ingestible device according to the present invention. [Figure 23] Figure 23 is a graph of the target standard curve for the dilution series for HIV quantification in an experiment according to an embodiment of the present invention. [Figure 24] Figure 24 is a graph of curves showing the typical operating range of a conventional PCR machine for amplifying target HIV-1. [Figure 25A] Figure 25A is a graph of curves showing the testing of a common bacterial pathogen, E. coli, in a system according to an embodiment of the present invention. [Figure 25B] Figure 25B is a graph in which a straight line is fitted between the quantitative cycle and the DNA copy number in the experiment shown in Figure 25A. [Figure 26] Figure 26 is a device diagram according to an embodiment of the present invention. [Figure 27] Figure 27 is a table of selected cardiovascular inflammatory biomarkers that can be determined, measured and / or monitored according to embodiments of the present invention. [Figure 28] Figure 28 is a table of selected diabetic inflammatory biomarkers that can be determined, measured and / or monitored according to embodiments of the present invention. [Figure 29] Figure 29 is a table of biomarkers selected for obesity, diabetes, and the progression of cardiovascular disease, which may be determined, measured, and / or monitored according to embodiments of the present invention. [Modes for carrying out the invention]

[0031] A description of exemplary embodiments of the present invention follows.

[0032] As used herein, the term "fluid" refers to both gases and liquids.

[0033] As used herein, the term “microfluidic” refers to a device and / or method that operates with a fluid volume of 0.1 to 100 μL, preferably between 1 and 10 μL, or with respect to a fluid volume of 0.1 to 100 μL, preferably between 1 and 10 μL.

[0034] As used herein, “fluid system” means a system through which a fluid flows, for example, a system through which a fluid flows in at least one channel, at least one chamber, at least one well and / or at least one port, each of which may be a macrofluid.

[0035] As used herein, “nucleic acid” refers to a macromolecule consisting of chains of monomeric nucleotides (polymers or oligomers). The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It will be further understood that the present invention can be used to detect and identify samples containing, in particular, peptide nucleic acids (PNA), morpholino, locked nucleic acid (LNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA). In various embodiments of the present invention, nucleic acids may be derived from various sources, such as bacteria, viruses, humans, and animals, and in particular from plants and fungi. The source may be a pathogen. Or, the source may be a synthetic organism. Nucleic acids may be genomic, extrachromosomal, or synthetic. Where the term “DNA” is used herein, those skilled in the art will understand that the methods and devices described herein may be applied to other nucleic acids, such as RNA or those mentioned above. Furthermore, the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” are used herein to encompass, but are not limited to, polymeric forms of nucleotides of any length, including, but not limited to, ribonucleotides or deoxyribonucleotides. No distinction in length is intended between these terms. Furthermore, these terms refer only to the primary structure of molecules. Therefore, in certain embodiments, these terms may include triple-stranded, double-stranded, and single-stranded DNA, PNA, and triple-stranded, double-stranded, and single-stranded RNA. These also include modifications such as methylation and / or capping, as well as the unmodified forms of polynucleotides.More specifically, the terms "nucleic acid," "polynucleotide," and "oligonucleotide" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), other types of polynucleotides that are N or C glycosides of purine or pyrimidine bases, and other polymers containing non-nucleotide backbone chains, such as polyamides (e.g., peptide nucleic acid (PNA) and polymorpholino (commercially available as Neugene from Anti-Virals, Inc., Corvallis, Oreg., USA)) polymers, as well as other synthetic sequence-specific nucleic acid polymers that contain nucleic acid bases in a configuration that enables base pairing and base stacking as found in DNA and RNA.

[0036] As used herein, “protein” refers to a biomolecule consisting of a chain of one or more amino acids. Proteins differ from one another primarily in the sequence of amino acids dictated by the nucleotide sequence of the gene that codes for them. A peptide is a single linear polymer of two or more amino acids linked by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues; if a chain contains multiple peptides, it may be called a polypeptide. Proteins consist of one or more polypeptides. Immediately after or during synthesis, protein residues are often chemically modified by post-translational modifications, which alter the physical and chemical properties, folding, stability, activity, and ultimately function of the protein. Proteins occasionally have non-peptide groups attached to them, which may be called prosthetic groups or cofactors.

[0037] As used herein, “biological sample” refers to any sample of material containing nucleic acids and / or proteins that can be extracted, analyzed, and detected. Preferably, the material is in liquid or gaseous form, or can be dissolved or suspended in a liquid or gas, or can be liquefied or made into a gaseous form, or otherwise prepared for analysis by the devices and methods of the present invention. Solid samples such as feces or soil samples can be loaded onto a chip, for example, in water. Aerosol biological samples may be used. “Biological sample” may be, or be a part of, a tissue sample, a biofluid sample, an environmental sample, or a sample of other types. Preferably, biological samples are derived from biofluids such as, but are not limited to, blood, saliva, semen, urine, amniotic fluid, cerebrospinal fluid, synovial fluid, vitreous fluid, gastric juice, nasopharyngeal aspirate, and / or lymph. Biological samples may also be materials or fluids contaminated with nucleic acid and / or protein sources. Biological samples may be tissue samples, water samples, air samples, food samples, or crop samples. Preferably, the biological sample analysis detects one or more of the following: waterborne pathogens, airborne pathogens, foodborne pathogens, or crop-borne pathogens.

[0038] As used herein, the term “biological analysis” refers to the performance of biochemical assays that use cells, nucleic acids, proteins, or other biomolecules as starting materials to extract information for the purposes of diagnosis, species identification, point mutation and / or disease susceptibility allele differentiation, qualitative analysis, quantitative analysis (e.g., determining viral load), and other purposes as taught herein. One example of the application of this technique is pathogen detection, which involves extracting genetic information from cells. As used herein, “steps” in “biological analysis” refer to steps of extraction, optionally amplification, and detection.

[0039] As used herein, “biomarker” refers to a measured characteristic that can be used as an indicator of a particular biological situation or state. For example, biomarkers may be measured and evaluated to examine normal biological processes, pathogenic processes, or pharmacological responses to therapeutic treatments.

[0040] As used herein, “therapeutic treatment” refers to the planned treatment of a health problem.

[0041] As used herein, “gene analysis unit” refers to a processor such as a hardware computer processor or other dedicated digital signal processor, which receives, stores, outputs, and processes electrical signals based on, for example, a computer program, where the electrical signals are related to gene analysis. For example, the gene analysis unit may be programmed to determine a biomarker based on an input signal related to a nucleic acid to be detected, and to determine and output a personalized genome profile based on that biomarker.

[0042] As used herein, “distribution control unit” refers to a processor such as a hardware computer processor or other dedicated digital signal processor, which receives, stores, outputs, and processes electrical signals based on, for example, a computer program, where the electrical signals relate to distributing at least a portion of a product. For example, a distribution control unit may be configured to determine at least a portion of a product to distribute based at least partially on a personalized genomic profile.

[0043] As used herein, “distribution activator” refers to a device or system configured to distribute at least a portion of a product. For example, a distribution activator may comprise at least a portion of one of the following: a vending machine; an automated counter; and a kiosk; and may comprise a three-dimensional printer configured to print at least a portion of a product or to distribute pre-packaged products.

[0044] A portion of an embodiment of the present invention, such as a gene analysis unit, may be implemented using one or more computer systems, for example, functioning on a computer processor and being part of program code. For example, an embodiment may be implemented using hardware, software, or a combination thereof. If implemented in software, the software code may be stored on any form of non-transient computer-readable medium, whether provided to a single computer or supplied between multiple computers, and may be loaded and executed on any suitable processor or array of processors.

[0045] General explanation As those skilled in the art will understand, the present invention may include hardware components, software components, and / or combinations of software and hardware components. In one embodiment, the present invention is a portable processing means interfaced with a compact integrated chip, which in one embodiment extracts, amplifies, and detects nucleic acids from a biological sample according to computer-readable instructions integrated into a computer-readable medium, and in another embodiment separates proteins, for example, by electrophoresis. The system may use interchangeable modules, as further described below in relation to Figure 2.

[0046] As used herein, the term “compact” refers to the design of a microfluidic device layout that minimizes the space used by fluid channels, chambers, wells, and ports, as achieved, for example, by a microfluidic device layout, as discussed below. In one embodiment, the use of intersecting fluid channels and shared wells is an example of a compact design. As used herein, the term “integrated” refers to the design of a microfluidic device layout in which fluid channels, chambers, wells, and ports used for various purposes are assembled on / within the same microfluidic device. For example, the chip embodiment shown in Figure 4 comprises a nucleic acid extraction chamber used for nucleic acid extraction, a nucleic acid amplification chamber used for nucleic acid amplification, and a detection channel used for separating and detecting nucleic acids. It should be understood that even if multiple chambers and channels are specified in the preamble, there may be “at least one” such chamber or channel. Furthermore, it should be understood that one or more “modules” used in the present invention may share a common chamber, for example, one or more of the extraction, amplification, and detection modules may share one or more common chambers. As used herein, the term “portable” refers to a system or device or mobile device that can be easily carried or transported by hand. As used herein, the term “mobile device” refers to a small, portable device that typically has a display screen along with touch input and / or a small keyboard, weighs less than approximately 10 kg, and includes, for example, smartphones, tablets, laptops, or other portable medical devices.

[0047] The system may store indicators of detected nucleic acids on a computer-readable medium or in the cloud, and compare these indicators with records stored in a genome or transcriptome database, which may be stored on a computer-readable medium or in a remote location. In an alternative embodiment, information about reference standards is stored in computer-readable memory located within the system. In yet another embodiment, nucleic acid standards are contained within an integrated chip that may be used with the system of the present invention.

[0048] The processor means may include, for example, a personal computer, microprocessor, or other suitable computer or smartphone, such as a minicomputer, microcomputer, UNIX machine, Intel processor, or similar device. It also typically includes conventional computer elements (not shown) such as a motherboard, central processing unit (CPU), random access memory (RAM), disk drive, and terminal devices such as a keyboard and display. The RAM stores an operating system such as Windows CE or another operating system, and appropriate software for processing signals related to detected nucleic acids or proteins.

[0049] Parts of the embodiments of the present invention described herein may be implemented using one or more computer systems. For example, embodiments may be implemented using hardware, software, or a combination thereof. When implemented in software, the software code may be stored on any form of non-transient computer-readable medium, whether provided to a single computer or supplied between multiple computers, and may be loaded and executed on any suitable processor or array of processors.

[0050] Furthermore, it should be understood that computers can be integrated into any of several forms, including laptop computers, tablet computers, or personal data assistants (PDAs), smartphones, or any other suitable portable or mobile electronic device, which may not normally be considered a computer but possess sufficient processing power, such as a computer integrated into a device.

[0051] Furthermore, a computer may have one or more input and output devices. These devices may, among other things, be used to provide a user interface. Examples of output devices that may be used to provide a user interface include a printer or display screen for the visual display of output, and a speaker or other sound-producing device for the audible display of output. Examples of input devices that may be used for a user interface include a keyboard, as well as pointing devices such as a mouse, touchpad, and digitizing tablet. In other examples, a computer may receive input information via speech recognition or through another visual format.

[0052] Such computers may be interconnected by one or more networks in any suitable form, including corporate networks or local area networks such as the Internet or wide area networks. Such networks may be based on any suitable technology and may function according to any suitable protocol, including wireless networks, wired networks or fiber optic networks.

[0053] Furthermore, the various methods or processes described herein may be coded as software executable on one or more processors using any one of various operating systems or platforms. Moreover, such software may be written using any of several suitable programming languages ​​and / or programming or scripting tools, and may be compiled as executable machine code and / or intermediate code that runs on a framework or virtual machine.

[0054] In this regard, at least a portion of the present invention may be embodied as a computer-readable medium (or a plurality of computer-readable media) (e.g., computer memory, one or more floppy disks, compact disks, optical disks, magnetic tapes, flash memory, field-programmable gate array circuit configurations or other semiconductor devices or other tangible computer storage media) coding one or more programs that perform methods for carrying out the various embodiments of the present invention when executed on one or more computers or other processors. The computer-readable medium or media(s) are transportable so that the programs or programs(s) stored therein can be loaded onto one or more different computers or other processors to carry out the various embodiments of the present invention described above.

[0055] In this regard, it should be understood that one implementation of the above embodiments includes at least one computer-readable medium on which a computer program (e.g., a set of instructions) is coded to perform some or all of the above functions of these embodiments when executed on a processor. As used herein, “computer-readable medium” includes only non-temporary computer-readable medium that can be considered a machine or manufacture (i.e., a product). A computer-readable medium may be, for example, a tangible medium on which computer-readable information can be coded or stored, a storage medium on which computer-readable information can be coded or stored, and / or a non-temporary medium on which computer-readable information can be coded or stored. Other, non-exclusive, examples of computer-readable mediums include computer memory (e.g., ROM, RAM, flash memory, or other types of computer memory), magnetic disks or tapes, optical disks, and / or other types of computer-readable medium that can be considered a machine or manufacture.

[0056] In this specification, the terms “program” or “software” are used in a general sense and refer to any kind of computer code or set of computer-executable instructions that can be used to program a computer or other processor to implement the various aspects of the present invention described above. Furthermore, according to one aspect of this embodiment, it should be understood that one or more computer programs that implement the method of the present invention when executed do not need to reside on a single computer or processor, but can be distributed in a modular manner among multiple different computers or processors to implement the various aspects of the present invention.

[0057] Computer-executable instructions can take many forms, such as program modules, which are executed by one or more computers or other devices. Generally, a program module includes routines, programs, objects, elements, and data structures that perform a specific task or implement a specific abstract data type. Typically, the functionality of program modules can be combined or distributed as desired in various embodiments.

[0058] In one embodiment, the portable assay system 10 is approximately the size of a mobile wireless device, as shown in Figure 1. The user interfaces with the portable assay system 10 using a display means 12 (e.g., a liquid crystal display) and an input means 14 (e.g., a keyboard). To use the portable assay system 10, the user places the sample 22 on the integrated chip 20 (described in more detail below) and inserts the integrated chip 20 into a slot 16 of the system itself.

[0059] Once the user loads the integrated chip 20, the portable assay system 10 extracts, amplifies, and detects nucleic acids from the sample 22 using the methods described below, or depending on other modules used, such as electrophoresis. A microprocessor (not shown) processes the detected signal, which may be shown to the user via the display means 12 and transmitted to other users via the transmission means 18. The transmission means 18 can be used to send and receive modulated data signals relating to the biological sample 22.

[0060] The term "modulated data signal" refers to a transmitted signal that encodes information within the signal by having one or more set or altered features. Examples of modulated data signals include carrier waves or other transmission mechanisms. The transmission medium can be any information transmission medium. Examples of transmission media include, but are not limited to, wired media such as wired networks or wired direct connections, and wireless media such as acoustic, infrared, radiation, microwave, spread spectrum, and other wireless media technologies.

[0061] The present invention may include a genome or transcriptome database storing multiple genome or transcriptome profiles or target biomarker sequences. In one embodiment, the present invention may include a signal profile of a single reference sample. The device of the present invention may also connect to geographically located genome or transcriptome databases, such as those stored at National Institutes of Health, Centers for Disease Control, etc. Such connections would facilitate tracking and regulating responses to disease outbreaks at widely dispersed analysis sites. Examples of analysis sites include hospitals, border checkpoints, airports, refugee camps, farms, quarantine zones, disease areas, homeless facilities, nursing homes, meatpacking plants, and food processing centers. Those skilled in the art will understand that there are many other analysis sites to which the present invention may be applicable.

[0062] The present invention does not require a direct connection to a genome or transcriptome database, but may require one if necessary. In other embodiments, the device may connect to a genome or transcriptome database via various public or private networks, such as a local area network (LAN), wide area network (WAN), or the internet. In one embodiment, the genome database is accessible via a public network such as the internet. The data is connected using secure means such as Secure Sockets Layer (SSL) or Secure Copy.

[0063] In the embodiment shown in Figure 1A, the portable assay system 10 connects to the genome database 5 via an internet connection 1 and a server 3. The processing unit 15 of the portable assay system 10 transmits and receives data to and from the genome database 5 and data stored in the memory 17 using a transmission unit 18, which may be an antenna, an Ethernet connection, or other means suitable for information transmission. The processing unit 15 controls the retrieval and processing of DNA data using extraction, amplification, and detection modules 40, 50, and 60. The processing unit 15 stores the retrieved data in the memory 17 and displays it to the user via a display unit 12; it also receives commands and queries from the user via an input unit 12.

[0064] In some versions of the present invention, the device may be mobile or point of use, and may be handheld.

[0065] Extraction-Amplification-Detection Module The portable assay system 10 (Figure 1) uses a combination of modules for extracting, amplifying, and detecting nucleic acids from the sample 22 (Figure 1). The total processing time, including data processing 70 for any of the detected signals, is typically less than 20 minutes, and may be less than 180 minutes, less than 120 minutes, less than 90 minutes, less than 60 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes, less than 5 minutes, or less than 1 minute, as shown in Figure 3.

[0066] As shown in Figure 2, each module may include one of the various implementations and combinations.

[0067] A biological sample 22 (Figure 1) is loaded onto the integration chip 20 (Figures 1 and 4). Loading of the biological sample can be achieved manually via the sample inlet port 100 (Figure 4) or via an automatic loading means in the portable system 10. For example, the inlet port 100 can be loaded with a manually or automatically operated loading device such as a pipette. Alternatively, in embodiments intended for field use, the inlet port 100 can be loaded directly with a cotton swab, or directly with a needle-pricked finger by pressing the finger against the inlet port 100. Capillary action causes blood to flow from the opening into the inlet port 100.

[0068] Nucleic acids are extracted by the nucleic acid extraction module 25 (Figure 4) on the integrated chip 20 under the control of the nucleic acid extraction control module 40 (Figures 2 and 3). For example, referring to Figure 2, the nucleic acid extraction control module 40 may comprise any suitable means for carrying out the methods listed below or any other suitable means for extracting nucleic acids from a biological sample: chemical extraction (WO 2005 / 05073691), sonication (WO 1999 / 9933559), mechanical shearing encompassing microfabricated protrusions on the integrated chip (U.S. Patent No. 5,635,358 and WO 2006 / 06029387), thermal means for disrupting cell membranes (see, for example, WO 2005 / 05011967), electroporation encompassing means for applying various voltages to a biological sample loaded on the integrated chip, wherein the voltage is sufficient to disrupt the cells of the biological sample (U.S. Patent Application Publication No. 2004 / 6783647), silica beads which may have nucleic acid probes in covalent bonds (WO 2005 / 05073691 and WO Magnetic beads having nucleic acid probes by covalent bonds (U.S. Patent Application Publication No. 2002 / 6344326), or 2003 / 03104774). All of the above references are incorporated by reference in their entirety. Preferably, in embodiments in which magnetic beads are used, magnetic particle capture means such as magnets are included in the nucleic acid extraction module 40.

[0069] In one embodiment, the magnetic beads may optionally have nucleic acid probes attached to them covalently. In this embodiment, the magnetic beads can be controlled in the extraction module using a small electromagnet or magnet. Possible magnetic beads to be used are any of the commercially available magnetic bead nucleic acid purification kits available from distributors such as Agencourt Bioscience, Cosmo Bio Co., Ltd., Invitek GmbH, Polysciences, Inc., Roche Applied Science, B-Bridge International, Dynal Biotech, Novagen, or Promega. The use of magnetic beads for DNA or RNA purification is described, for example, in Caldarelli-Stefano et al., "Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR", Mol Pathol. 1999 June; 52(3): 158-160.

[0070] As shown in Figure 2A, the extraction control module 40 may include a control unit 250, a power supply 252, and an electromagnet 254. Upon receiving a command from the extraction control module 40, the control unit 250 applies power from the power supply 252 to the electromagnet 254 and applies a magnetic field (not shown) to the extraction module 25. As a result, the magnetic beads (not shown) of the extraction module 25 cluster along the inner wall of the extraction chamber (not shown) of the extraction module 25. The control unit 250 releases the magnetic beads by stopping the operation of the power supply 252, thereby stopping the application of the magnetic field by the electromagnet 254.

[0071] The extracted nucleic acids can be amplified within the nucleic acid amplification module 27 (Figure 4) of the integrated chip 20 under the control of the nucleic acid amplification control module 50 (Figures 2 and 3). Amplification can be achieved using any suitable amplification technique, including the conventional PCR techniques disclosed in U.S. Patent No. 4,683,202 and U.S. Patent No. 4,683,195, both of which are incorporated herein by reference in their entirety. The nucleic acids can also be amplified using a constant temperature technique, such as the technique taught in U.S. Patent No. 7,494,791, which is also incorporated herein by reference in its entirety.

[0072] In some embodiments, nucleic acid amplification includes reverse transcription polymerase chain reaction (RT-PCR).

[0073] Referring to Figure 2, the nucleic acid amplification control module 50 may comprise any suitable means for performing nucleic acid amplification (i.e., increasing the number of copies of the nucleic acid template). The amplification may be proportional or exponential. In one embodiment, module 50 includes means for well-based nucleic acid amplification. As shown in Figure 2B, such means may include means 201 for heating / cooling the nucleic acid amplification module 27 of the integrated chip 20. Examples of heating means 201 for heating / cooling the nucleic acid amplification module of the integrated chip include Peltier devices (WO 1998 / 9850147, the whole thereof incorporated herein by reference) and thin-film based devices. In other embodiments, means for heating the nucleic acid amplification module of the integrated chip (see Figure 4, element 25) may include infrared heating means (WO 1996 / 9641864, the whole thereof incorporated herein by reference) or microwave radiation heating means.

[0074] As shown in Figure 2B, the heating means 201 may include a heating / cooling element 203, a temperature sensor 205, a processing unit 207, a power control unit 209, and a power supply 211. Upon receiving a command from the amplification control module 50, the processing unit 207 initiates heating / cooling by instructing the power control unit 209 to supply power from the power supply 211 to the heating / cooling element 203. The processing unit 207 monitors the temperature of the heating / cooling element 203 by means of the temperature sensor 205 and adjusts the command to the power control unit 209 as necessary to maintain the desired temperature with respect to the amplification module 27.

[0075] In other embodiments, module 50 (Figures 2 and 3) includes means for fluid-based nucleic acid amplification (see, for example, U.S. Patent No. 7,041,481, which is incorporated herein by reference in its entirety). Such means may include fluid flow generating means for generating a flow of buffer through the nucleic acid amplification module of the integrated chip, the fluid flow generating means functionally coupled with temperature control means for controlling the temperature of the nucleic acid amplification module of the integrated chip.

[0076] In other embodiments, module 50 (see Figures 2 and 3) includes means for real-time polymerase chain reaction (PCR) control means for performing nucleic acid amplification (see, for example, U.S. Patent No. 7,315,376, which is incorporated herein by reference in its entirety).

[0077] In other embodiments, module 50 (see Figures 2 and 3) includes means for applying controlled tension to nucleic acid strands within the nucleic acid amplification module of the integrated chip (see Figure 4, element 25). Details of such means are provided in U.S. Patent No. 7,494,791, which is incorporated herein by reference in its entirety.

[0078] As used herein, the term “tension,” when used in the context of nucleic acid amplification, processing, and / or detection, refers to thermal cycling or thermal denaturation of double-stranded nucleic acids, or to non-thermally driven processing of nucleic acid amplification or denaturation, or to alternative means to annealing or primer extension. The application of “tension” to nucleic acids is the result of applying a physical force to the nucleic acid chain that is not solely derived from thermal energy. Tension may be “precisely controlled” (as defined elsewhere herein) and / or controlled to be adjustable and / or variable.

[0079] The precisely controlled tension may be mechanical tension, hydrodynamic tension, electromagnetic tension, or a combination thereof. Furthermore, in some embodiments, the means for applying the precisely controlled tension to the nucleic acid chain is configured to operate at a constant temperature. As used herein in the context of nucleic acid amplification, processing, and detection, the term “constant temperature” refers to a nucleic acid amplification method that does not require any thermal cycling, and preferably an amplification process in which the entire process can be carried out at substantially the same temperature.

[0080] Embodiments using means for controlled tension on nucleic acid strands within an integrated chip nucleic acid amplification module (see Figure 4, element 25) offer significant advantages over conventional (thermal cycling) methods for nucleic acid amplification. These advantages include overall greater accuracy, and particularly when amplifying "difficult" sequences (e.g., GC-rich sequences), improved sequence length, reaction yield, and reaction rate (total time of amplification reaction). Other significant advantages include the ability to improve amplification efficiency and amplification fidelity by, for example, inducing calibration exonucleases through the use of tension (see teachings in U.S. Patent Nos. 7,494,791, 8,632,973, and U.S. Patent Application No. 14 / 106,399, all of which are incorporated herein by reference).

[0081] Embodiments employing means for applying controlled tension to nucleic acid chains include a mechanism for applying variable and controlled amounts of tension to nucleic acid molecules held within a nucleic acid amplification module of an integrated chip (see Figure 4, element 25). Such a mechanism may comprise a first surface and a second surface having means for fixing nucleic acid molecules thereon, where the first and second surfaces are configured to move relative to each other. Alternatively, such a mechanism may comprise at least one surface having means for fixing nucleic acid molecules thereon, and the device further comprises a mechanism for providing controlled and variable fluid flow over the nucleic acid molecules. In yet another embodiment, such a mechanism may comprise at least one surface having means for fixing nucleic acid molecules thereon, and further comprises fluid flow passages distributed between the means for fixing the nucleic acid molecules. In other embodiments, such a mechanism may comprise a mechanism for providing controlled and variable fluid flow over the nucleic acid molecules configured to create a velocity gradient in the laminar fluid flow. Occasionally, such a mechanism may comprise a fluid flow channel, a stagnation point in the fluid flow, a back-propagating fluid flow, or a combination thereof, configured to provide a velocity gradient in the laminar fluid flow. In other embodiments, such a mechanism may comprise a series of optical, electrical, or magnetic manipulators (e.g., optical tweezers, individually controllable magnetic beads) configured to manipulate particles bound to nucleic acid molecules. Means for retaining nucleic acids within the nucleic acid amplification module of the integrated chip (see Figure 4, element 25) may include activatable primers containing complexing groups for immobilizing the extension product obtained during nucleic acid amplification. Alternatively, nucleic acid polymerase may be immobilized or otherwise retained on the surface of the integrated chip 20.

[0082] The amplified nucleic acid is detected by the nucleic acid detection module 29 (Figure 4) on the integrated chip 20 under the control of the nucleic acid detection control module 60 (Figures 2 and 3).

[0083] In various embodiments, module 60 may include fluorescence or electro-optic detection means 950 for detecting nucleic acids, as shown in Figure 9A. The fluorescence detection means 950 may include a light emitter 952, such as a light-emitting diode and / or laser diode; a data acquisition device 954, such as a photodetector, photomultiplier tube (PMT), or charge-coupled device (CCD); an avalanche light-emitting diode; and a processing unit 956 for storing and processing the acquired data. In one embodiment, the light emitter 952 excites a sample with emitted radiation 960, producing a fluorescence signal 962 that is sensed by the data acquisition device 954. In the embodiment shown in Figure 9A, the emitted radiation 960 irradiates the detection module 29 with a transmitted geometry, although it should be understood that reflected and other geometries are also used. The data acquisition device 954 converts the fluorescence signal 962 into a regulated data signal 958, which is sensed and recorded by the processing unit 956.

[0084] The amplified nucleic acids can be separated and detected using capillary electrophoresis disclosed in U.S. Patent No. 6,261,431, which is incorporated herein by reference in its entirety. In some embodiments, module 60 may include capillary electrophoresis (CE) control means for performing nucleic acid separation, the CE control means functionally coupled to fluorescence detection means. The CE control means may further include a high-voltage control unit for applying a voltage to the entire nucleic acid detection module 60 of the integrated chip 20, the voltage being sufficient to perform nucleic acid separation.

[0085] Referring to Figure 4, in one embodiment, CE is carried out in detection channel 112, which may be filled with a suitable buffer, and may also be pre-filled with a sieving matrix such as agarose, hydroxypropylcellulose, or polyacrylamide. In certain embodiments, the end user may fill detection channel 112 (Figure 4) with a sieving matrix at the time of use.

[0086] In an alternative embodiment, the nucleic acid detection control module 60 further comprises a hybridization microarray, such as a GeneChip® microarray available from Affimetrix, Santa Clara, CA.

[0087] In one embodiment, the nucleic acid detection control module 60 comprises electrochemical means for detecting nucleic acids. An example of electrochemical means for detecting nucleic acids is provided in U.S. Patent Application Publication 2008 / 0081329, which is incorporated herein by reference in its entirety. Briefly, such means carry out a method for determining the presence or absence of a target substrate in a test sample. The means includes an electrode having a conductive surface and a probe bonded to the conductive surface and capable of binding to a target substrate. The probe bonded to the conductive surface comes into contact with the test sample and forms a surface-bound target complex. The surface-bound target complex further comprises a first redox complex. The surface-bound probe or the surface-bound target complex comes into contact with a fluid medium containing a second redox complex, if present, where one of the first and second redox complexes is a redox transition metal complex capable of carrying out a redox reaction, and the other of the first and second redox complexes is a redox catalyst complex capable of catalyzing the redox reaction of the redox transition metal complex. Redox transition metal complexes do not undergo any significant redox reactions in the absence of redox catalyst complexes. Redox reactions of redox transition metal complexes catalyzed by redox catalyst complexes are detected.

[0088] In another embodiment, the nucleic acid detection control module 60 comprises impedance measuring means for detecting nucleic acids. An example of impedance measuring means for detecting nucleic acids is described, for example, in U.S. Patent No. 7,169,556, which is incorporated herein by reference in its entirety. Briefly, the method involves contacting a nucleic acid having a first part and a second part with an oligonucleotide attached thereto, the oligonucleotide located between a pair of electrodes having a sequence complementary to the first part of the nucleic acid sequence, and the contact occurs under conditions effective for enabling hybridization of the oligonucleotide on the substrate with the nucleic acid. After hybridization of the nucleic acid and oligonucleotide has occurred, the nucleic acid bound to the substrate is contacted with a first type of conductive particle (e.g., metal beads), the conductive particle being made of an electrically conductive material and having one or more types of oligonucleotides attached thereto, at least one of the types of oligonucleotides having a sequence complementary to the second part of the nucleic acid sequence, and the contact occurs under conditions effective for enabling hybridization of the oligonucleotide on the substrate with the nucleic acid and for the conductive particle to form a complexed test substrate. After the second hybridization occurs, the test substrate is brought into contact with a salt solution of a salt concentration effective in sufficiently dehybridizing and removing nonspecifically bound conductive particles. Hybridization / dehybridization results in a detectable change in inter-electrode impedance due to the presence of specifically bound conductive particles.

[0089] According to embodiments of the present invention, any one of many possible techniques for extraction, amplification, and detection may be used in combination; or, in practice, one or more of extraction, amplification, and detection may be omitted from the combination of extraction, amplification, and detection. For example, in the modular system of Figure 2, any one of the possible types of modules shown under extraction module 40 may be used for extraction, or other types of extraction modules may be used; such extraction module 40 may be used in combination with any one of the possible types of modules shown under amplification module 50 or another type of amplification module; furthermore, such extraction module 40 and amplification module 50 may be used in combination with any one of the possible types of modules shown under detection module 60 or another type of detection module. Different combinations of such modules may be used depending on the target to be detected. Firstly, biological samples such as blood, saliva, etc. may be used as the biological sample to be analyzed; or solid samples (e.g., fecal samples) or aerosol samples may be used. In extraction module 40, the target nucleic acid or protein is separated from the rest of the biological sample. Amplification can then occur in the amplification module 50. In another example, if the target is a protein, it can be passed directly to a detection module 60, such as a capillary electrophoresis module or one of the other detection modules shown in Figure 2, or to other types of detection modules, such as a melting curve analysis module, a chemiluminescence module, a quantum dot module, a module using nanoparticles such as gold nanoparticles, or a detection module based on the use of radiation. In yet another example, if the first biological sample to be analyzed is nucleic acid, it may not be necessary to use the extraction module 40, and the sample can be sent directly to the detection module 60.With respect to the amplification module 50, in addition to the module types shown in Figure 2, other possible modules that can be used are Loop-Mediated Isothermal Amplification (LAMP) modules, Helicase-Dependent Amplification (HDA) modules, SDA (Strand Displacement Amplification) modules, or bridge amplification modules. LAMP techniques, such as those described in T. Notomi, et al., Nucleic Acids Research, 28, e63 (2000), may be used; HDA techniques, such as those described in Vincent M, Xu Y, Kong H. (2004), "Helicase-dependent isothermal DNA amplification," EMBO Rep 5 (8): 795-800, may be used; and SDA techniques, such as those described in GT Walker, et. al., Proc. Natl. Acad. Sci USA, 89, 392-396 (1992), may be used; all of these references are incorporated herein by reference. The system modules may be interchangeable between thermal and constant temperature amplification modules; any of the amplification techniques taught herein, such as Helicase-Dependent Amplification (HDA) and Strand Displacement Amplification (SDA), as well as any of the techniques taught in U.S. Patent Nos. 7,494,791, 8,632,973, and U.S. Patent Application No. 14 / 106,399, all of which are owned by Nanobiosym, Inc., and the entire teachings of these references are incorporated herein by reference. In one specific embodiment, a portable assay system 10 comprises an extraction control module 40 configured to use magnetic beads for nucleic acid extraction, an amplification control module 50 using a thermal cycling method using a Peltier device, and a detection control module 60 for separating and detecting nucleic acids using capillary electrophoresis and fluorescence signal detection.

[0090] According to embodiments of the present invention, different integrated chips may be analyzed by a single mobile device, and each possible different integrated chip performs a different function that the single mobile device recognizes and performs. For example, one chip may be used for nucleic acid analysis, and another chip may be used for protein analysis by the same mobile device.

[0091] Figure 3 shows a diagram illustrating the general flow of materials and data through the system of the present invention, such as the module system shown in Figure 2. The extraction, amplification, and detection modules 40, 50, and 60 are typically operated in about 4 minutes each, compared to several hours to several weeks using conventional techniques. In embodiments according to the present invention, detection can occur in less than 2 hours, less than 1 hour, less than 30 minutes, less than 1 minute, or less than 1 second from the start of sample analysis. In one embodiment, the integrated chip 20 housing the extraction, amplification, and detection modules 40, 50, and 60 typically weighs about 35 g, while the application-specific integrated circuit (ASIC) used to process the detection signal weighs about 25 g, resulting in a total cartridge weight of about 60 g. The modules are located on the integrated chip and controlled by a corresponding control module (not shown) of the portable assay system 10.

[0092] Similarly, in some embodiments, the nucleic acid amplification chamber 108 (Figure 4) can be heated and cooled using a Peltier device, a thin-film heater, or other temperature control means, while a thermocouple monitors the temperature of the chamber 108. The capillary electrophoresis process can be controlled by a detection control module that controls the strength and duration of the electric field applied across the entire channel used for capillary electrophoresis.

[0093] In some embodiments, system 10 (Figure 1) may include means for generating fluid pressure to move biological samples and / or nucleic acids through the integrated chip. Fluid pressure may be generated by a peristaltic pump, piston pump, pneumatic pump, or any other suitable means. Alternatively, pressure may be applied to microfluidic channels within the chip using a manual pump syringe connected to a suitable inlet port, e.g., sample inlet port 100, reagent addition port 108, sample well 120, buffer well 116, or buffer waste well 114 (Figure 4).

[0094] Means for controlling valves (shown in Figures 7 and 8A and 8B, and referred to as the valves discussed below) may be incorporated into system 10 (Figure 1). The valves control the flow of fluid between different modules and along various transport channels. Any number of valve technologies may be used, including passive plug valves, mechanically activated valves, electromagnetically activated valves, magnetic fluid valves, pressure valves, or any other suitable valve.

[0095] In some embodiments, system 10 (Figure 1) includes data processing means for storing, classifying, quantifying, and transmitting data obtained from the analysis of biological samples. The data processing means may include means for wireless data transmission.

[0096] In certain embodiments, the signal intensity over time can be analyzed to provide an indicator of the type and amount of nucleic acid species present in the biological sample 22 (Figure 1). For example, by plotting the detected signal against the signal produced by the reference sample, the same pathogen will produce signal peaks at the same point in time (assuming the same analytical conditions), allowing the user to determine whether pathogens present in the reference sample are present in the test sample. The data processing means further includes a virus load calculation means for calculating the virus load (for example, by integrating the area showing the fluorescence signal under the curve as a function of time).

[0097] In other embodiments, the system 10 further includes data display means for outputting data.

[0098] The portable assay system 10 is interfaced with the integrated chip 20 via electrical, chemical, and mechanical interfaces. Clamps and / or fasteners securely fix the chip in place to prevent displacement of electrodes, detectors (such as photodetectors), or heaters due to vibration. Heaters (optionally cooling elements such as inkjet coolers) (not shown), located below, above, or on the amplification control module 40, heat and / or cool specific parts of the chip as follows. Similarly, electrodes located near the detection control module 60 can be used to control separation and fluid flow through the chip as follows. Since the chip 20 is transparent, electromagnetic beams, such as infrared or electromagnetic beams for heating, or optical beams for fluorescent tag identification, can be used to explore the wells, chambers, and channels of the chip 20.

[0099] Integrated chip The following description refers to specific embodiments of the integrated chip 20 (Figures 1 and 4), but it should be understood that other chip designs may be used in conjunction with the portable assembly system 10 (Figure 1) of the present invention.

[0100] In one embodiment, the present invention is a compact integrated chip for use with a system for rapid analysis of biological samples.

[0101] The integrated chip 20 may be formed from glass, but not limited to polyethylene, polypropylene, poly(urethane-imide), poly(tetrafluoroethylene), polycarbonate, cyclic olefin copolymer (COC), polyamide, cyclic olefin polymer (COP), poly(methyl methacrylate), polyacrylamide, polystyrene, PMMA, or any other suitable material or combination of materials having good optical properties.

[0102] The integrated chip comprises: a nucleic acid extraction module comprising at least one sample inlet port for injecting biological samples and reagents; a nucleic acid extraction module comprising at least one extraction chamber for extracting nucleic acids from a biological sample, wherein the extraction chamber is connected to the sample inlet port by at least one sample transport channel; a nucleic acid amplification module comprising at least one amplification chamber for amplifying nucleic acids, wherein the nucleic acid amplification chamber is connected to the extraction chamber by at least one nucleic acid transport chamber; and a nucleic acid detection module comprising at least one detection channel for separating and detecting nucleic acids, wherein the detection channel is connected to the nucleic acid amplification chamber by at least one amplification product transport channel. Preferably, at least one sample inlet port, at least one extraction chamber, at least one amplification chamber and at least one detection channel are arranged to minimize space usage. As discussed above in relation to Figure 2, interchangeable modules may be used.

[0103] Preferably, the nucleic acid amplification module further includes at least one reagent addition port fluid-connected to at least one nucleic acid transport channel. In certain embodiments, the nucleic acid detection module further includes at least one sample well fluid-connected to at least one amplification product transport channel. In some embodiments, the nucleic acid detection module further includes at least one sample discard well fluid-connected to at least one detection channel; a buffer discard well fluid-connected to at least one detection channel; and a buffer well fluid-connected to at least one detection channel. Preferably, the integrated chip of the present invention includes at least two sample inlet ports; at least two nucleic acid extraction chambers, each connected to one of the at least two sample inlet ports by its respective sample transport channel; at least two nucleic acid amplification chambers, each connected to one of the at least two extraction chambers by its respective nucleic acid transport channel; and at least two detection channels, each connected to each nucleic acid amplification chamber by its respective amplification product transport channel.

[0104] In some embodiments, at least two detection channels intersect. In one embodiment, the detection channels 112 intersect at a right angle, as shown in Figure 4. It is understood that any angle of intersection can be selected depending on the typical general shape of the integrated chip 20, the number of detection channels, and the desired layout of the integrated chip. A compact layout is preferred. Referring to Figure 4, the intersection of the detection channels 112 provides the advantage of a compact design, where the sample discard well 118 and the buffer well 116 can be shared by multiple (or all) of the detection channels 112. Furthermore, an embodiment in which the user loads molecular sieves into the detection channels 112 and intersects the detection channels 112 in the buffer well 116 provides the advantage of loading all detection channels simultaneously.

[0105] The integrated chip may further comprise at least two reagent addition ports, each fluidly connected to a nucleic acid transport channel; and at least two sample wells, each fluidly connected to a amplification product transport channel. Preferably, at least two sample wells are configured for use in adding and / or disposing of further reagents.

[0106] In certain embodiments, the integrated chip further comprises at least one sample waste well, each connected to at least two detection channels by at least two sample waste channels. Preferably, at least one sample waste well is configured for use for adding and / or disposing of further reagents.

[0107] In other embodiments, the integrated chip further includes at least one buffer waste well fluid-connected to at least two detection channels; and a buffer well fluid-connected to at least two detection channels. Preferably, at least one buffer waste well and the buffer wells are configured for use in adding and / or disposing of further reagents.

[0108] In one embodiment, a buffer well is located at the intersection of at least two detection channels, where the buffer well is in fluid communication with the intersecting detection channels.

[0109] Preferably, each sample discard channel is coupled with a detection channel downstream of at least one amplification product transport channel, thereby effectively increasing the cross-sectional area of ​​the intersection of at least one amplification product transport channel and at least one amplification product transport channel (see Figures 8A–8C and the following description). As used herein, the term “downstream” is defined as the direction in which nucleic acids pass through the detection channel during separation.

[0110] In some embodiments, the integrated chip further includes microfabricated projections (posts) positioned in at least one nucleic acid extraction chamber and / or at least one sample transport channel. As described above, the microfabricated projections positioned on the integrated chip may be used for mechanical shearing of cells in a biological sample, for example, as described in U.S. Patent No. 5,635,358 and WO 2006 / 06029387, which are incorporated herein by reference in their entirety.

[0111] The integrated chip may further comprise at least two electrodes for applying voltage across at least one sensing channel. In other embodiments, the integrated chip may include additional electrical contacts for providing power and / or control signals to an electrically activated mechanism that may be located within the integrated chip.

[0112] In one embodiment, the present invention is a novel valve assembly shown in Figures 5 and 6A and 6B, which are described in further detail below. Thus, in one embodiment, the present invention is a valve assembly in a microfluidic device.

[0113] In one embodiment, the valve assembly comprises a microfluidic channel for fluid transport, formed between an upper and lower surface and having a longitudinal axis; a valve port on the upper surface for receiving a passive plug; and a passive plug configured for insertion into the valve port. Preferably, the portion of the lower surface opposite the valve port has a uniform depth along the longitudinal axis. This arrangement simplifies the manufacturing process of the integrated chip by eliminating or reducing the strictness required for component placement.

[0114] In another embodiment, the valve assembly comprises a transport channel for transporting fluid; a magnetic fluid channel intersecting the transport channel; a first magnetic fluid reservoir and a second magnetic fluid reservoir, both connected by the magnetic fluid channel; magnetic fluid in one or both of the magnetic fluid reservoirs; and a permanent magnet or electromagnet for filling the magnetic fluid channel with magnetic fluid. The magnetic fluid is typically a colloidal mixture containing magnetic particles suspended in a liquid, further comprising a detergent / surfactant mixed in the liquid to prevent the particles from agglomerating together (see Berger, et al. (July 1999). "Preparation and properties of an aqueous ferrofluid". Journal of Chemical Education 76 (7): pp. 943-948), which is incorporated herein by reference in its entirety). Any commercially available magnetic fluid may be used, such as the magnetic fluid available from Ferrotec Corporation, Bedford, NH.

[0115] In yet another embodiment, also with reference to Figures 7A and 7B, the present invention relates to a valve assembly in a microfluidic device comprising: a reservoir; an inlet transport channel for transporting fluid having an output end connected to the reservoir; first and second outlet channels for transporting fluid, each having an input end connected to the reservoir; magnetic fluid placed in the reservoir; and a magnet for controlling the position of the magnetic fluid in the reservoir. During operation of the valve assembly in Figures 7A and 7B, the magnetic fluid in the reservoir is positioned to close either the output end of the inlet channel; or the input end of the first outlet channel instead of the input end of the second outlet channel; or the input end of the second channel instead of the input end of the first outlet channel; or to not close the output end of the inlet channel or the input ends of the first and second outlet channels.

[0116] In the embodiment shown in Figure 4, the integrated chip 20 comprises eight sample regions 130, each comprising a nucleic acid extraction module 25, a nucleic acid amplification module 27, and a nucleic acid detection module 29. The chip may be made of glass, acrylic, or any other suitable material. Typically, the chip is about the size of a thick credit card. In one embodiment, the chip has a length of 1 to 4 inches and a width of 1 to 4 inches; preferably, the integrated chip 20 is about 2 inches wide and 4 inches long.

[0117] The nuclear sample region 130 includes one sample inlet port 100, one extraction chamber 104, one amplification chamber 108, and one detection channel 112. For each biological sample, it is understood that there is a separate, identifiable pathway from each sample inlet port to each extraction chamber, then to each amplification chamber, and then to each detection channel. In this way, cross-contamination of samples is avoided, and sample integrity is ensured. However, there are common wells shared by multiple sample regions (e.g., a sample waste well 118 and a buffer well 112). This sharing reduces the space required for the integrated chip layout and also reduces material waste.

[0118] Assuming that the size of the sample region 130 is sufficient for extracting, amplifying, and detecting nucleic acids, it is understood that the chip can be configured with any number of sample regions (e.g., 1, 12, 24, 256, 386, or 512). Given that appropriate references are available, the sample region 130 can be used to analyze multiple different samples relating to the same target biomarker sequence or pathogen pair, one sample relating to multiple different pathogens or biomarkers, or any appropriate order or combination of samples and pathogens or biomarkers.

[0119] The biological sample 22 (see Figure 1), reagents used for extraction, amplification, and detection, move between different wells and reaction chambers through microfluidic transport channels 102, as shown in Figure 4. In specific embodiments, the transport channels 102 have a cross-sectional area of ​​10 to 1000 μm × 10 to 1000 μm, preferably 200 × 100 μm, and are large enough to accommodate the flow of magnetic beads used in the extraction process embodiment. In one embodiment, ports and wells such as the sample inlet port 100, reagent inlet / outlet port 108, buffer waste well 114, buffer well 116, sample waste well 118, and sample well 120 have a diameter of 0.5 to 10.0 mm, preferably 0.9 to 5.0 mm, and their depth is determined by the thickness of the integrated chip 20. The nucleic acid amplification chamber 108 typically has a diameter of 0.1 to 30.0 mm, preferably 10.0 mm, and a depth of 10 μm to 19.0 mm, preferably 0.5 mm. The nucleic acid extraction chamber 104 may be 1 to 25 mm in length and 1 to 30 mm in width, preferably 15 mm in length and 10 mm in width. The nucleic acid detection module 29 of the integrated chip 20 using capillary electrophoresis has a detection channel 112 having a cross-sectional area of ​​10 to 200 × 10 to 200 μm, more preferably 50 × 10 μm.

[0120] The fluid flow through the channels, ports, wells, and chambers is controlled by a combination of valves and propulsion means, which may be a syringe of a manual pump connected to the sample inlet port 100. Other propulsion means, such as peristaltic pumps and piston pumps, may also be used. The propulsion means pushes the fluid through the channels and chambers, as long as a closed valve does not obstruct the fluid flow. The valve may use one of several technologies, including a manually activated plug, a mechanically activated plug, an electromagnetically activated plug, a magnetic fluid valve, a pressure valve, or any other suitable technology.

[0121] For example, Figure 5 shows a valve assembly 152 comprising a passive plug valve 150, a valve port 151, and a transport channel 102 formed by an upper surface 160 and a lower surface 161. The passive plug valve 150 can be inserted into the valve port 151 to control the flow of fluid into the nucleic acid amplification chamber 108 via the transport channel 102. The valve 150 can be inserted manually or activated mechanically. In one embodiment, the inlet sample well 100, reagent inlet / outlet port 106, sample waste well 118, sample well 120, buffer waste well 114, and buffer well 116 can act as valve ports 151 to receive the passive plug valve 150.

[0122] Figures 6A and 6B illustrate an alternative method for controlling fluid flow using a ferrofluid valve 600. A ferrofluid is typically a colloidal mixture containing magnetic particles suspended in a liquid, further comprising a detergent / surfactant mixed into the liquid to prevent the particles from agglomerating together (see also Berger, et al. (July 1999). "Preparation and properties of an aqueous ferrofluid". Journal of Chemical Education 76 (7): pp. 943–948, the whole of which is incorporated herein by reference). Any commercially available ferrofluid may be used, such as the ferrofluid available from Ferrotec Corporation, Bedford, NH. The inline ferrofluid valve 600 comprises a ferrofluid channel 601 positioned across the transport channel 102. One end of the ferrofluid channel 601 terminates in a ferrofluid reservoir 604 filled with ferrofluid 602, and the other end terminates in a waste reservoir 606. In the "open" state, the magnetic fluid 602 remains in the magnetic fluid storage area 604, allowing the fluid to flow through the transport channel 102 in the fluid flow direction 608, as shown in Figure 6A. By placing the magnet 610 near the waste storage area 606, the magnetic fluid 602 is attracted through the magnetic fluid channel 601, blocking the transport channel 102, as shown in Figure 6B.

[0123] Figures 7A and 7B illustrate how the magnetic fluid valve 600 can be used to control the flow of fluid into or out of a multi-port chamber and well. In the embodiments shown in 7A and 7B, the fluid enters the well 702 through the inlet 704. A small amount of magnetic fluid 602 remains in the well 702. With both outlets 706a and 706b open, the magnetic fluid 602 remains stationary in a stable position inside the well 702, as shown in Figure 7A. The well 702 shown in Figures 7A and 7B has one inlet port 702 and two outlet ports 706a, 706b; in other embodiments, the well 702 may have multiple inlet and outlet ports 704, 706.

[0124] The fluid enters the well 702 through the inlet 704. Depending on the position of the magnetic fluid 602, the fluid can exit the well 702 through either the outlet port 706a or the outlet port 706b. As shown in Figure 7B, the magnetic fluid 602 may be positioned to block outlet 706a using a magnet 610, leaving the other outlet 706b open for fluid release. Similarly, outlet 706b may be blocked while outlet 706a is open. It should also be understood that the inlet 704 may be blocked. It should also be understood that the direction of fluid flow may be reversed. In one embodiment, different fluids may enter the well 702 through outlets 706a and 706b, while the inlet 704 may serve as a fluid discharge port.

[0125] Referring again to the embodiment shown in Figure 4, in one embodiment of the present invention, the user loads magnetic beads coated with streptavidin (e.g., Applied Biosystems FMAT® Streptavidin Beads, 6-8 microns) into the sample area 130. The magnetic beads may move along the microfluidic transport channel 102 to the nucleic acid extraction chamber 104 under the pressure of a syringe pump or other suitable fluid pressure generating means. Alternatively, the magnetic beads may already be loaded into the extraction chamber 104. The user then loads a biological sample 22 into the sample area 130 via the sample inlet port 100. Once the biological sample 22 is loaded, it moves from the sample inlet port 100 to the extraction chamber 104 through the same transport channel 102. The magnetic beads (not shown) adhere to the nucleic acids of the biological sample 22 by streptavidin coating. Once the adhesion process is complete, applying a magnetic field to the extraction chamber causes the magnetic beads (and the attached nucleic acids) to move in the direction of the magnetic field, thereby extracting nucleic acids from the biological sample 22.

[0126] The user completes the extraction process by flushing the extraction chamber 104 with a washing solution (not shown) injected through the sample inlet port. The washing solution flows through the extraction chamber 104 to the reagent inlet / outlet port 106, from which the user extracts the washing product. The user may repeat the washing cycle until the extracted nucleic acid is sufficiently free of mixture for amplification and detection.

[0127] Once the extracted nucleic acid no longer contains the mixture, it is pushed downstream into the nucleic acid amplification chamber 108 (for example, by applying pressure with a syringe or pump), and the user then loads reagents into the nucleic acid amplification chamber 108 via the sample inlet / outlet port 106. The user then isolates the amplification chamber by closing the appropriate valve before initiating amplification by conventional amplification techniques. The reagents in the amplification chamber 108 may be heated and cooled using a thermoelectric heater (not shown). In one embodiment, the reagents include primers, DNA polymerase (such as Taq polymerase), deoxynucleotide triphosphate, buffer solution, and divalent cations.

[0128] Once the nucleic acid has been sufficiently amplified, the valve is opened to isolate the amplification chamber 108, and the amplified nucleic acid is flowed through the transport channel 102 to the sample well 120 connected to the detection channel 112, as shown in Figure 8A.

[0129] In one embodiment, capillary electrophoresis is used to detect and identify nucleic acids present in a biological sample 22. Capillary electrophoresis involves passing an electric current through an electrolyte, such as an aqueous buffer solution mixed with the test sample, within a short channel (approximately 50 μm in length), such as a detection channel 112. The current causes the sample to move downstream through the detection channel; however, the electrophoretic rate of the compounds depends, among other variables, particularly molecular weight, current, and channel size, so the compounds separate as they move.

[0130] In one embodiment, the user may add electrolyte to the detection channel 112 through a buffer well 116 (also shown in Figure 4) and add a sieving matrix. In embodiments where the buffer well 116 is connected to multiple detection channels 116 as shown in Figure 4, simultaneous loading of all detection channels is achieved. Once the buffer well 116 is filled, an electric field 130 applied to electrodes positioned on or near the sample well 120 and the sample waste well 118 causes the amplified nucleic acid 23 to migrate from the sample well 120 to the sample waste well 118 via the detection channel 112, as shown in Figure 8B. Typically, the electric field 130 is approximately 400V. The extracted nucleic acid accumulates in an accumulation region 113 located within the detection channel 112 between the intersection of the transport channels 102 connecting the sample well 120 and the sample waste well 118 to the detection channel 112. The waste from the sample well 120 continues to move to the sample waste well 118.

[0131] Figure 8C shows how applying an electric field 131 from buffer well 116 to buffer waste well 114 across the entire detection channel 112 causes separation of the amplified nucleic acid 23 sample into species 140 according to their molecular weight. Typically, the strong electric field 131 is approximately 6 kV. Different target biomarker sequences or unit replication sequences of different pathogens have different molecular weights, so different biomarkers or pathogens move downstream of the detection channel 112 at different rates. Unknown species can be identified by comparing their rate to that of a known reference (not shown) in the same detection channel 114 or in the same detection channel 112 under the same electric field strength 131.

[0132] Figure 9B shows an exemplary capillary electrophoresis detection control module 60. As described above, the electric field 131 attracts the species 140 downstream of the detection channel 112 toward the buffer waste well 114. As the species 140 moves downstream of the detection channel 112, they pass through the measurement area 1005 one by one, where the species 140 are examined by a laser beam 1004 or an LED or other light source to produce a signal that can be interpreted to indicate a target biomarker sequence or an indicator of the type of pathogen and to quantify the corresponding load.

[0133] In one embodiment, the fluorescence signal 1010 is produced by irradiating a separated load 140 with a laser beam 1004, an LED, or another suitable frequency light source. The laser 1002 produces the laser beam 1004, which can be directed to the measurement area using a mirror 1006. Alternatively, a lens can be used to focus the laser beam 1004 onto the sample. The beam may also be produced by a light-emitting diode or another light source instead of a laser, and may or may not be connected via an optical fiber cable. It will be understood that various optical arrangements can be used to inspect species 140.

[0134] The laser beam 1004 excites the separated load 140 to produce a fluorescent beam 1010, which is separated from the laser beam 1004 by a dichroic beam splitter 1007. In one embodiment, the detector 1012 generates a photocurrent (not shown) in response to the intensity of the fluorescent beam 1010. The photocurrent is sent to a processor 1014, which may be used to analyze the detected fluorescent beam 1010. For example, the analysis may include comparing the detected signal with a reference signal trace 1021 of a known reference sample in the output signal graph 1020, where matching peaks in the trace indicate the presence of the same biomarker sequence or pathogen. Integrating the area under the peaks yields an index of the relative amount of biomarker or pathogen (i.e., viral load) present in each sample. In an embodiment, software correlates the output signal graph 1020 with the reference signal trace 1021 using standard correlation techniques to produce an output suitable for user interpretation.

[0135] Lab-on chip for nucleic acid or protein detection Embodiments of the present invention include an integrated chip capable of performing the above-described biological assays on a single device, also known as an integrated microfluidic chip or lab-on-a-chip. The integrated chip is designed to sequentially perform the following three processes: (1) extraction from living cells; (2) sequence-specific nucleic acid amplification by PCR; and (3) size separation of the amplified DNA by capillary electrophoresis combined with fluorescence detection of the separated DNA.

[0136] It will be understood that integrated microfluidic chips or lab-on-a-chips similar to those discussed in the current section can be used for protein detection, along with techniques appropriately modified for protein detection applications. For example, in protein detection, in one embodiment, only an extraction module and a detection module without amplification may be used. Furthermore, in some embodiments, only a detection module may be used. In other embodiments, only the extraction, nucleic acid sequencing, and detection modules may be used without an amplification module. Any one of these modules may be used individually or in combination.

[0137] In one embodiment, the invention described herein is an integrated chip for rapid extraction, amplification, and separation of nucleic acids from biological samples. The invention further includes a hardware system that receives at least one integrated chip. The integrated chip is a microfluidic device comprising microfluidic channels fluidically connected in sequence to functional modules: a nucleic acid extraction module, a nucleic acid amplification module, and a nucleic acid separation module.

[0138] In one embodiment, a target biological sample is loaded onto an integrated chip, and nucleic acids are extracted from the biological sample in an extraction module. For example, the biological sample may be cells, and cellular DNA is extracted from the cells. The extraction process preferably utilizes nucleic acid precipitation using magnetic beads. The extracted nucleic acids are then transported by fluid pressure to a nucleic acid amplification module, where they are amplified using a polymerase chain reaction. In some embodiments, thermal cycling is used for amplification. In other embodiments, amplification utilizes, but is not limited to, constant-temperature amplification techniques encompassing the teachings of Nanobiosym, Inc., all of which are incorporated herein by reference. The amplified nucleic acid product is then transported by fluid pressure to a nucleic acid separation module, where the nucleic acids are separated and detected using capillary electrophoresis. Preferably, the nucleic acid product is detected using fluorescence detection with fluorescent nucleic acid primers. The three sequential processes described above occur on the same integrated chip.

[0139] The sequential processes of extraction, amplification, and separation / detection are controlled by a hardware system including a nucleic acid control module, a nucleic acid amplification control module, and a nucleic acid detection control module. Preferably, each module controls: (1) the application of magnets for precipitation of nucleic acids with attached magnetic beads, (2) thermal cycling during amplification of the extracted nucleic acids, and (3) fluorescence-assisted detection of nucleic acids.

[0140] Referring to Figure 10, the present invention enables advances in healthcare and other industries in various embodiments. The integrated chips and hardware systems of the present invention, described in detail below, can be used for the analysis of biological samples in biomedical testing in biomedical applications encompassing biological defense, environmental testing, foodborne pathogen testing, medical services, organ transplantation, life science research, industrial applications, agriculture, forensic testing, veterinary medicine, and public health applications including public emergencies.

[0141] Compact integrated chip design Referring again to Figure 4, Figure 4 illustrates an integrated microfluidic chip 20 according to an embodiment of the present invention, having eight sample regions 130, each having an extraction module 25, an amplification module 27, and a separation module 29. On the integrated chip 20, the above processing is achieved in closed chambers, open wells, and interconnected channels, and the combination of these closed chambers, open wells, and interconnected channels constitutes the microfluidic chip. In this integrated chip 20, the multiplex design accommodating the analysis of multiple samples is achieved by a structure in which each sample has an independent fluid path in a separate sample region 130 from input to final detection, with no possibility of cross-contamination. Each sample region 130 includes one sample inlet port 100, one extraction chamber 104, one amplification chamber 108, and one detection channel 112. There are common wells shared by multiple sample regions 130 (e.g., a sample discard well 118 and a buffer well 116). Sharing this common well reduces the space required for integrated chip layouts and also reduces waste of materials and reagents such as capillary electrophoresis running buffers.

[0142] In the specific embodiment shown in Figure 4, four sample input wells 100 (loading ports) are located at each end of the tip, thus accommodating eight samples. Each of these wells 100 is then connected by a channel 102 to a set of closed extraction chambers 104 designated for nucleic acid extraction. Each of these extraction chambers 104 is then connected to another set of open wells (reagent inlet / outlet ports 106), allowing fluid to flow through the extraction chambers 104 as necessary to carry out the extraction assay. In a preferred embodiment, the fluid flow through the channels, ports, wells, and chambers is controlled by a combination of valves and propulsion means, as follows: Suitable propulsion means include a manual pump syringe, peristaltic pump, or piston pump connected to the sample input wells 100.

[0143] The reagent inlet / outlet port 106 then connects to another set of closed amplification chambers 108, designed for amplification. The amplification chambers 108 also connect to the sample well 120 on one side and to the reagent inlet / outlet port 106 (shared with the extraction chamber 104, respectively) on the other side, again allowing fluid to flow through the amplification chambers 108.

[0144] Finally, each fluid path ends in an independently separate set of separation / detection channels (CE channels) 112, and electrophoretic separation is achieved towards the endpoints of the CE channels 112 before fluorescence detection. The electrophoresis module is again uniquely designed to multiplex the injection and separation of all eight samples that have already undergone extraction and amplification processing on-chip.

[0145] The well 120 on the electrophoresis side of the amplification chamber 108 is open and used as the CE sample well 120. Thus, each sample (or amplification product) has a separate CE sample well 120 connected to each amplification chamber 108. However, the buffer well 116 required for the CE assay is shared between samples (either all samples or between two adjacent sample pathways), as shown in Figure 4. This multiplex design also reduces the complexity of high-voltage wiring: by relying on a common buffer well 116, one electrode can be shared by multiple channels, thus requiring fewer electrodes to operate the system. For example, a single high-voltage (preferably greater than 6kV) electrode can be used for all eight samples subjected to CE. The CE operation of tip 20 ensures that the shared buffer well 116 does not pose a risk of sample cross-contamination. The CE operation, including loading and unloading of wells 114, 116, 118, and 120, and the application of the electric field will be discussed in more detail below with reference to Figures 4 and 8A–8C.

[0146] Typically, the chip 20 is about the size of a thick credit card. In one embodiment, the chip 20 has a length of 2.5 cm to 25 cm, a width of 2.5 cm to 15 cm, and a thickness of 0.1 mm to 10 mm; preferably, the integrated chip 20 is about 5 cm wide, 10 cm long, and 2 mm thick. Those skilled in the art will understand that the specific size and design of the chip are a matter of design preference.

[0147] In a preferred embodiment, the characteristic dimensions of the tip 20 are 10–200 μm × 10–200 μm (e.g., 50 μm × 20 μm) with respect to the detection (CE) channel 112, and 10–1000 μm × 10–1000 μm (e.g., 200 μm × 100 μm) with respect to all other channels. With respect to the channels through which the magnetic beads flow, it will be understood that the channel dimensions are selected to ensure an unobstructed flow of the magnetic beads. The detection (CE) channel 112 may be 10–120 mm in length (e.g., 80 mm). Larger detection CE channel sizes require higher voltages for CE analysis. The size of the transport channel 102, as well as other channels such as the extraction chamber 104 and reagent inlet / outlet port 106, the reagent inlet / outlet port 106 and amplification chamber 108, the amplification chamber 108 and sample well 120, and the channel connecting the sample well 120 and detection channel 112, are not critical.

[0148] In preferred embodiments, circular wells and chambers, such as buffer well 116, have a diameter of 5 to 15 mm (e.g., 10 mm) and a depth of 0.05 to 1 mm (e.g., 0.5 mm). Hexagonal chambers, such as extraction chamber 104, may have a length of 5 to 25 mm (e.g., 15 mm) (at tapered ends) and a width of 5 to 15 mm (e.g., 10 mm). This type of hexagonal design facilitates easy fluid flow in extraction chamber 104, especially since the processing involves the flow of large-volume fluids. Other embodiments may accommodate circular chambers with a diameter of 0.1 to 30 mm and a depth of 0.01 to 19 mm, and hexagonal chambers with a length of 1 to 25 mm (at tapered ends) and a width of 1 to 30 mm.

[0149] Preferred embodiments of the access wells and inlet ports, such as the inlet well 100, have a diameter of 0.9 to 5 mm, and their depth is determined by the thickness of the plastic used to make the tip 20. Alternative embodiments may accommodate access wells with a diameter of 0.5 to 10 mm. Again, the depth of the well is determined by the thickness of the plastic used, and is typically 0.5 to 2 mm.

[0150] Fabrication of compact integrated chips Embodiments of microfluidic chips fabricated for the purposes and applications described herein are manufactured from plastics, polymers, or biodegradable polymers; therefore, these chips are cost-effective to manufacture and can be used as disposable devices. The disposable form thus ensures that problems such as lingering contamination, which are common with the reuse of biological processing tools, are minimized to an almost negligible degree. These chips may also be fabricated from glass, silicon, and other materials.

[0151] The manufacturing process associated with these chips involves microfabrication of a structure in silicon (Si), glass, aluminum (Al), or other such material to create a master having the desired microfluidic structure. This creates a negative microfluidic structure on the microfabricated Si / glass device. This process then involves forming a rubber-based mold on the microfabricated Si / glass device to form a positive mold. Hot embossing of the rubber mold onto a plastic plaque forms a microfluidic structure identical to that of the initial Si / glass microfabricated device. In this process, the plastic plaque is heated and extruded onto the positive rubber mold, making the plaque malleable. This malleability causes the plastic of the plaque to conform to the pattern of the rubber mold, creating a negative structure on the plastic plaque identical to the negative structure of the initial Si / glass microfabricated device. The plastic plaque, with the negative structure embossed on it, is finally perforated and bonded to another untreated plastic plaque to create a closed fluidic structure.

[0152] For embossing and bonding, plastic materials such as polyethylene, polypropylene, poly(urethane-imide), poly(tetrafluoroethylene), polycarbonate, polyimide, cyclic olefin copolymers (COC) and cyclic olefin polymers (COP), poly(methyl methacrylate), polyacrylamide, and polystyrene, or other such materials, can be used, depending on the customization required by the specific application.

[0153] The hot embossing process described above for manufacturing plastic microfluidic chips enables the production of low-cost devices suitable for disposable use. Furthermore, since plastic manufacturing can typically be scaled up and down in a non-linear cost ratio, batch processing of these microfluidic devices can significantly reduce manufacturing costs. The following biological assays can be performed using the manufactured plastic microfluidic chips.

[0154] Module design Referring to Figure 4, the chip 20 includes three modules: a DNA extraction module 25, a PCR amplification module 27, and an electrophoretic separation module 29 for biological analysis or diagnostic assays. Typically, the entire process of extraction, amplification, and separation occurs within the same chip, and the sample is transported by a fluid flow from one module to the next.

[0155] This integrated process includes cell lysis, binding of magnetic beads to nucleic acids, resuspension / elution of the nucleic acid-attached beads, amplification of the extracted nucleic acids, and detection of the amplified nucleic acids. During the extraction process, the sample is incubated and washed to produce extracted DNA in extraction chamber 104. For amplification, which takes place in amplification module 27, the extracted DNA is loaded into amplification chamber 108 together with the amplification master mix. The DNA and amplification master mix are sealed in amplification chamber 108 and heated and cooled according to an appropriate thermal protocol. Once the protocol is complete, the amplified sample is transferred to sample well 120 for sequential separation and detection. A sieving matrix / gel, buffer and optionally molecular size standards are loaded into appropriate CE wells and buffer wells (wells 114, 116, 118, and 120), and a first electric field is applied to allow the sample to enter detection (CE) channel 112. By applying a second electric field to the detection channel 112, the sample is separated into species, which then propagate beyond the detection region, where fluorescent tags attached to the species can be stimulated and detected. Extraction, amplification, and detection are described in further detail below.

[0156] Nucleic acid or protein extraction module It will be understood that extraction modules similar to those discussed in this section can be used for protein extraction, along with the following nucleic acid technologies, which have been appropriately modified for use in protein extraction.

[0157] The nucleic acid extraction method incorporated into this integrated chip utilizes magnetic beads, where chemically dissolved cellular nucleic acids are captured by the magnetic beads. The magnetic beads can be coated with any suitable ligand that binds to the isolated target. Examples of coatings include streptavidin (for use with biotin-labeled targets), antibodies against the desired target, protein A, protein G, and oligo-dT (for use with mRNA).

[0158] These beads may optionally be held in a chamber for subsequent direct amplification from the DNA-attached beads, or by eluting the DNA from the beads before amplification. To perform the extraction assay, first, a combination of lysis buffer and streptavidin-coated magnetic beads (e.g., available from Dynal Direct®, available from Invitrogen, or prepared by the end user according to a protocol well known in the art) is mixed with the cell sample, and the extraction process is carried out according to the following protocol. The components and specifications of the buffer and beads used in the extraction protocol are also described in detail in the following protocol.

[0159] An exemplary protocol for the extraction procedure for use with a compact integrated chip, encompassing the chamber 20 shown in Figure 4, includes: cell lysis, binding of magnetic beads to nucleic acids, resuspension / elution of the nucleic acid-bound beads, and detection of nucleic acids. Loading approximately 0.01–10 μL of cell sample into the on-chip extraction chamber via the sample input well 100 (see Figure 4) can be performed using a pipette or syringe.

[0160] Next, the magnetic beads / solution are loaded into the extraction chamber containing the previously dispensed cell sample via well 100. Dispensing the bead solution into the extraction chamber induces mixing of the cells and bead solution as it flows.

[0161] As used herein, the term “lysis buffer” refers to any buffer used for the purpose of lysing cells for use in experiments analyzing cellular compounds. Typically, lysis buffers include surfactants that cause cell membrane disruption. Examples of such surfactants include Triton X-100, NP40, or Tween-20. Other components of lysis buffers may include protease inhibitors, DNA-degrading enzymes; disulfide reducing agents such as magnesium salts, lysozyme, and β-mercaptoethanol; ion chelating agents such as EDTA; and preservatives such as sodium azide. Those skilled in the art will understand that the specific buffer used is determined based on the starting material and the target to be analyzed.

[0162] Once the extraction chamber 104 is filled, the loading of the magnetic beads stops. The filled extraction chamber is incubated at room temperature for several minutes to allow a target, such as DNA, to bind to the ligand coated on the magnetic beads.

[0163] In some embodiments, once incubation is complete, a magnet is placed (engaged, if an electromagnet is used) below the extraction chamber 104 (Figure 4) at the tip 20. The magnet settles the magnetic beads and allows them to be retained in the extraction chamber 104 as cell debris is washed out. Typically, the magnet used exerts a field of about 12,000–13,000 gauss in the extraction chamber. It will be understood that the strength of the magnet depends on the size of the beads, the size of the sample, and other factors and can be adjusted accordingly.

[0164] In some embodiments, the crude extract is washed out of the extraction chamber while the magnet is still engaged. The washing solution is loaded into the same on-tip well as the previous loading. Examples of washing solutions include Tris / EDTA(TE) buffer, phosphate buffer (NaH2PO4, NaCl, pH 8), or other standard washing buffers known in the art.

[0165] In some embodiments, the washing step may be repeated a sufficient number of times to ensure that the target to be purified (e.g., DNA) is free from the lysis solution and cell debris. Those skilled in the art will readily be able to determine the number of washing cycles required to achieve this. The captured target molecule (e.g., DNA) can be eluted in the extraction chamber 104 using a standard elution buffer, as is well known in the art.

[0166] The solution flowing through the extraction chamber 104 is removed by pipette, syringe, pump, or any other suitable means through the reagent inlet / outlet port 106 on the other end of the extraction chamber. This waste solution is periodically removed through the reagent inlet / outlet port 106.

[0167] DNA / RNA amplification can be carried out in several different ways. One method involves directly adding magnetic beads, known not to inhibit amplification, to the amplification master mix along with the DNA / RNA bound to the beads. In other embodiments, DNA / RNA detection may be performed directly without amplification, for example, by directly reading the nucleic acid sequence.

[0168] As used herein, the term “master mix” refers to a solution comprising a polymerase (e.g., Taq DNA polymerase, reverse transcriptase), magnesium chloride, and a mixture of deoxyribonucleotides (dNTPs) in a reagent buffer (e.g., an aqueous solution of (NH4)2SO4, TrisHCl, Tween-20, pH 8.8). Those skilled in the art will understand that a particular master mix is ​​selected based on the target to be amplified, the test conditions, and the preferred polymerase.

[0169] The second method involves eluting DNA or RNA from beads and then resuspending the DNA in any standard buffer known in the art that does not inhibit amplification.

[0170] When nucleic acid-bound beads are used directly for amplification, the master mix is ​​loaded into the amplification chamber 108. The magnet below the extraction chamber 104 is then disengaged to allow the magnetic beads to float freely in the extraction chamber. To prevent the magnetic beads (and the DNA bound to the beads) from flowing out of the extraction chamber 104, the ports 106 and wells 120 on either side of the extraction chamber 104 are sealed. For example, the reagent inlet / outlet ports 106 and wells 120 can be sealed with plug valves, or the channel 102 connecting the inlet / outlet ports 106 and wells 120 to the extraction chamber 104 can be sealed using an elastic or magnetic fluid valve. In this way, the beads can move freely within the extraction chamber 104 but are prevented from leaving it. The extraction is complete and the chip is ready for amplification.

[0171] If DNA-bound beads are not used, the extraction chamber is flushed with resuspension buffer. The resuspension buffer is loaded into the extraction chamber 104 until it is completely filled again, and the wash buffer is moved out of the extraction chamber 104 and unloaded. The magnet at the bottom of the extraction chamber 104 is then disengaged, and the wells 100 and the reagent inlet / outlet ports 106 are sealed with appropriate valves.

[0172] Next, the chip is heated to 50–90°C (e.g., 70°C) on a Peltier device or another suitable heat source commonly used as a means of controlling or regulating temperature, such as a temperature control method for amplification. This induces the elution of DNA from the magnetic beads.

[0173] After heating, a valve opens that blocks the transport of fluid from the extraction chamber 104 to the amplification chamber 108 via the transport channel 102. Finally, the extracted DNA / RNA / protein flows from the extraction chamber 104 to the amplification chamber 108.

[0174] Once the DNA / RNA / protein extraction process is complete, and whether or not the DNA is bound to magnetic beads, the extracted DNA is ready for on-chip amplification, as detailed below. Depending on the volume of DNA / RNA solution (i.e., the concentration of DNA) required for amplification or detection, a portion of the eluted DNA / RNA may be unloaded from the extraction chamber and an amplification master mix may be added.

[0175] Amplification module As discussed elsewhere in this specification in accordance with embodiments of the present invention, the amplification module may be isothermal or thermal. Similarly, as discussed elsewhere in this specification in accordance with other embodiments of the present invention, the amplification module may be replaced by a single cycle of amplification or DNA / RNA replication or DNA / RNA sequencing, which may be isothermal or thermal. In another embodiment, no amplification is performed at all.

[0176] In one embodiment, the DNA / RNA extracted from the extraction module and the amplification master mix are mixed in the amplification module 108. The amplification master mix includes a set of sequence-specific forward and reverse primers that amplify the target gene sequence (usually a species-specific feature) with the extracted DNA / RNA (except for other standard reagents described later).

[0177] In some embodiments, a desired amount of amplification master mix is ​​loaded into the amplification chamber 108, and a thermal cycle is performed at a desired DNA denaturation temperature (70–100°C) and primer annealing temperature (40–70°C).

[0178] In some embodiments, amplification is achieved by arranging an integrated chip together with means of precise temperature control and, for example, a programmed temperature cycling protocol (see, for example, the protocol for amplification of a 700 bp DNA fraction characteristic of E. coli DH10B cells, as described in the Exemplary Section). Those skilled in the art can easily determine the number of cycles based on the starting concentration of the sample and the desired concentration of the final amplified product.

[0179] In other embodiments, amplification is achieved by arranging the integrated chip together with means for precise control of DNA or RNA molecules and, for example, a programmed tension cycling protocol.

[0180] In one embodiment, amplification is performed using a hardware module, which is described in detail below.

[0181] In certain embodiments, those skilled in the art would add 10°C to the above temperature and an additional minute to each incubation time.

[0182] Upon completion of the thermal or tension cycle, the DNA / RNA sequence is amplified as designed by the primers, and the sample is ready for analysis using the method described below.

[0183] Capillary electrophoresis (CE) module In certain embodiments, after the amplification / replication of a specific DNA sequence is complete, the replicated / amplified DNA molecules are subjected to size separation in the separation module 29 of the chip 20 shown in Figure 4, and then subjected to fluorescence detection. Electrophoresis-based detection is achieved as follows:

[0184] Referring again to Figure 4, a polyacrylamide sieving matrix (also referred to herein as the “gel”) (e.g., GenceScan polymer, ABI, or a homemade polyacrylamide solution suitable for use in capillary electrophoresis) is loaded into the detection (CE) channel 112. This is achieved by simultaneously distributing the sieving matrix from the central buffer well 116 on the integration chip 20 to all eight multiple separation modules 29. Next, CE running buffer (any standard buffer known to those skilled in the art suitable for use in capillary electrophoresis) is loaded into all but one of the CE wells (i.e., the buffer is loaded into CE buffer well 116, CE buffer waste well 114, and CE sample waste well 118, but not into CE sample well 120). The amplification product is first loaded into the CE sample well 120 by pressurizing the sample from the amplification chamber 108 into the CE sample well 120. The running buffer is then added to the amplification product in the CE sample well 120.

[0185] Those skilled in the art will understand that different gel media can be used for electrophoretic separation. For example, polyacrylamide or agarose gels (e.g., different concentrations from 0.1% to 20%) can be used for DNA and RNA separation. Sodium dodecyl sulfate (SDS) polyacrylamide gels or agarose gels can be used for protein separation. In protein analysis, low concentrations of acrylamide can be used to separate high molecular weight proteins (e.g., 5% for 40–200 kDa), while high concentrations of acrylamide can be used to separate low molecular weight proteins (e.g., 15% for 10–40 kDa). Similarly, low concentrations of agarose can be used for the separation of high molecular weight nucleic acids (e.g., 0.5% for 1–30 kbp), and high concentrations of agarose can be used for the separation of low molecular weight nucleic acids (e.g., 1.5% for 0.1–2 kbp).

[0186] Examples of buffers suitable for use in capillary electrophoresis include Tris / borate / EDTA (TBE) buffer (e.g., for nucleic acids separated on agarose matrix) and Tris / glycine / SDS (e.g., for proteins separated on polyacrylamide gel). Those skilled in the art will understand that the choice of running buffer depends on the target being analyzed.

[0187] Alternatively, if the size of the amplified product is to be confirmed using a size standard, the size standard (e.g., Gene Scan, TAMRA, or ABI) can be added to the amplified product, and the running buffer can be loaded on top. The running buffer and the amplified DNA can be mixed to form a homogeneous suspension in well 120. In this case, primers labeled with fluorophores similar to the size standard (e.g., VIC, ABI) can be used to label the amplified DNA, as can be predicted by the amplification process, in order to enable fluorescence detection of the DNA.

[0188] Any molecular weight standard suitable for nucleic acid separation can be used. Examples include the GelPilot® molecular weight marker available from QiaGen and the Molecular Weight Ladder available from New England Biolabs.

[0189] Fluorescently labeled nucleic acid primers are commercially available (e.g., from Sigma-Aldrich) or can be synthesized by those skilled in the art according to known protocols (see, for example, Prudnikov et. al. Nucleic Acids Res. 1996 November 15; 24(22): 4535-4542 or protocols available online at http: / / info.med.yale.edu / genetics / ward / tavi / n_label.html; the entire teachings of both publications are incorporated herein by reference). Examples of fluorescent labeling include fluorescein, 6-carboxyfluorescein, 5'-tetrachlorofluorescein, Texas Red (sulfolodamine 101 acid chloride), quantum dots, and fluorescently tagged nanoparticles.

[0190] Figures 8A–8C illustrate the CE treatment performed in the detection (CE) channel 112. Once the reagent is loaded into wells 114, 116, 118, and 120, a platinum electrode grid (not shown) is lowered onto the tip 20 (Figure 4), immersing the electrode end in all wells 114, 116, 118, and 120. As shown in Figure 8B, an electric field 130, such as a relatively low injection voltage (e.g., approximately 400V), is applied between the sample well 120 and the sample waste well 118 for a short time. This causes the amplified product (amplified nucleic acid 23) to move along the transport channel 102 and the detection (CE) channel 112 connecting the sample well 120 and the sample waste well 116. Next, as shown in Figure 8C, a strong electric field 131, such as a high voltage of approximately 6kV, is applied between the buffer well 116 and the buffer waste well 114. This leads to the movement of the amplified nucleic acid 23, captured in transport channel 102 and detection (CE) channel 112, along the pathway connecting these wells.

[0191] The application of a strong electric field 131 causes separation of DNA molecules based on their size. Further downstream from the initial position of DNA in the CE channel 112, in the detection channel 112 (for example, 75 mm from the point where DNA 23 is trapped between the buffer well 116 and the buffer waste well 114), fluorescence can be stimulated by the separated species 140 being appropriately irradiated with a laser source, an LED, or another light source with a wavelength matched to the excitation wavelength of the fluorophore attached to the species 140. Those skilled in the art can easily determine the appropriate wavelength to be used based on the selected fluorophore.

[0192] As described below with reference to Figure 11, the detection control module 406 of the hardware module 400 senses the fluorescence of excited fluorophores tagged with amplified DNA or a size standard.

[0193] The separation module 29 of chip 20 (Figure 4) can be conveniently used as a standalone module for several other applications, including DNA, RNA, and protein analysis. To create a chip tailored to a specified application, it may be necessary to manipulate combinations of flow parameters to obtain the desired level of analytical data (i.e., high resolution in separation).

[0194] In any embodiment described herein, the length of the detection channel 112 can be adjusted from several centimeters to several meters to accommodate the separation of a large number of independent molecules. This improves the ability to resolve the separated molecules.

[0195] Similarly, the applied electric fields (e.g., electric field 130 and strong electric field 131) can vary from millivolts to several thousand volts to achieve the desired level of fractionation (DNA / RNA / protein) separation. This improves the ability to resolve the separated molecules. Those skilled in the art can determine the applied electric field based on the length of the detection channel 112 (Figure 4), the desired resolution, and the target molecule.

[0196] Those skilled in the art will understand that different gel media can be used for electrophoretic separation. For example, polyacrylamide or agarose gels (e.g., different concentrations from 0.1% to 20%) can be used for DNA and RNA separation. Sodium dodecyl sulfate (SDS) polyacrylamide gels or agarose gels can be used for protein separation. In protein analysis, low concentrations of acrylamide can be used to separate high molecular weight proteins (e.g., 5% for 40–200 kDa), while high concentrations of acrylamide can be used to separate low molecular weight proteins (e.g., 15% for 10–40 kDa). Similarly, low concentrations of agarose can be used for the separation of high molecular weight nucleic acids (e.g., 0.5% for 1–30 kbp), and high concentrations of agarose can be used for the separation of low molecular weight nucleic acids (e.g., 1.5% for 0.1–2 kbp).

[0197] Furthermore, non-optical detection strategies, such as electrochemical detection strategies, can be used, mitigating the need to fluorescently label molecules detected by separation.

[0198] Kit and pre-loaded integrated chips In exemplary embodiments, different integrated chips may be prepared for the analysis of different pathogens, diseases, and biological samples.

[0199] End users of the integrated chip described herein can analyze various biological samples by arranging the chip and hardware system (described later) at various locations, as described above with reference to Figure 10. To further enable this capability, the integrated chip of the present invention may be included in a kit further comprising components such as reagents containing various buffers, molecular markers, molecular weight standards, magnetic beads, oligonucleotides, fluorescent dyes, CE sieving matrices, etc. Alternatively, many of these components may be pre-loaded onto the integrated chip.

[0200] Referring to Figure 4, an integrated chip 20 having at least one extraction chamber 104 may be fabricated, for example, including magnetic beads that may be coated with streptavidin. In some embodiments, at least one extraction chamber 104 may contain reagents for cell lysis and nucleic acid extraction (e.g., the lysis buffer described above). In some embodiments, at least one extraction chamber 104 may include microfabricated projections located in and / or within at least one transport channel 102, which may help mechanically shear and lyse cells in a biological sample. At least one amplification chamber 108 may be preloaded with magnetic beads, optionally fluorescently labeled oligonucleotides, amplification master mix, etc. At least one detection channel 112 may be preloaded with a CE sieving matrix and / or running buffer and / or molecular weight standards. In another embodiment, at least one detection channel 112 may include a hybridization microarray for identifying amplified nucleic acid targets.

[0201] In other embodiments, the integrated chip of the present invention may include at least two electrodes for applying a voltage across the entire area of ​​at least one sensing channel 112. In other embodiments, the integrated chip may include at least one fluid control valve for controlling the flow of fluid through at least one microfluidic channel. The valve may be a passive plug and may be one of the valve systems described below.

[0202] Precision control Embodiments according to the present invention utilize nanoengineering and precision control of molecules at the nanoscale. By precisely controlling molecular or fluid systems with ultra-high precision, embodiments according to the present invention can dramatically improve the overall quality produced by chemical and biochemical reactions carried out under highly controlled and tunable conditions on a nanoengineered or precision-engineered chip. Thus, by utilizing nanotechnology, embodiments according to the present invention can not only improve at least one step of the integrated process but also dramatically improve the overall signal-to-noise ratio, increasing the ability to detect trace amounts of biological targets such as pathogenic nucleic acids, biomarker sequences, or proteins in fluid samples. The precision control elements of nanoengineered chips and platform devices according to embodiments of the present invention tighten the overall Gaussian spread within products produced by on-chip reactions, enabling orders of magnitude improvements across several key performance metrics, including speed, accuracy, scalability, adaptability, and toughness.

[0203] Embodiments according to the present invention enable precise or nanoscale control of nucleic acids and proteins. Nanoscale or precision reactors enable unexpected improvements in the time, cost, speed, and infrastructure required to perform the analysis of biological samples compared to conventional larger-scale systems. For example, tension can be applied to nucleic acids, such as by an electromagnetic field acting in the reaction chamber, which can cause nucleic acids, such as a 2 nm wide DNA molecule, to stretch in a given direction. This increases the rate at which the reaction tends to occur, for example by opening the DNA. In this way, small-scale molecular control is achieved. Such small-scale control enables the achievement of a larger signal-to-noise ratio, improved yields, and reduces the time to equilibration in the reaction chamber. Nanoscale control can be achieved using a variety of forces, such as mechanical forces, electromagnetic forces, and / or hydrodynamic forces, and the use of the types of tension taught in U.S. Patent No. 7,494,791, the entire disclosure of which is incorporated herein by reference.

[0204] As used herein, the term “precise control” of a parameter refers to the ability to control the parameter so that repeated measurements of the parameter under unchanging conditions yield the same results within a range of precision, and further, the ability to adjust the parameter to a new value within such a range of precision. For example, according to embodiments of the present invention, a parameter controlling at least one step of a reaction used to analyze a biological sample can be precisely controlled to be within a range of plus or minus 10%, plus or minus 1%, plus or minus 0.1%, plus or minus 0.01%, plus or minus 0.001%, or plus or minus 0.0001%, and may be adjustable to a new value within such a range of precision. Such precision control can be carried out in numerous ways, such as by utilizing a controlled fluid flow or by applying tension, and includes precision control with respect to both proteins and nucleic acids.

[0205] As used herein, applying tension to nucleic acids or proteins involves applying a force directly or indirectly to the nucleic acids or proteins that has a tendency to stretch or elongate them. For example, tension can be applied to nucleic acids or proteins by direct application of mechanical tension, hydrodynamic stress in a fluid flow, or an electromagnetic field, whether acting on the nucleic acid or protein molecule itself and / or on a surface, substrate, or particle to which the nucleic acid or protein is bound.

[0206] According to embodiments of the present invention, the tension that tends to stretch the nucleic acid may be applied for at least one cycle. Furthermore, the tension that tends to stretch the nucleic acid may be applied and may change between each cycle of the following steps (a) to (e) in the nucleic acid amplification method: (a) Contacting one or more template strands of a single-stranded nucleic acid with one or more oligonucleotide primers that are complementary to one or more portions of the template strands; (b) Annealing at least one of the primers to the portion of one or more template chains that is complementary to the primer; (c) Contacting one or more template chains with nucleic acid polymerase and at least four different creoside triphosphates; (d) extending at least one annealed primer with nucleic acid polymerase, thereby forming one or more extension products bound to one or more template chains; (e) Separating one or more extension products derived from one or more template chains; and, (f) Amplifying nucleic acids by repeating steps (a), (b), (c), (d) and (e), Here, at least one of the one or more extension products of step (e) is used as a template chain in the next cycle of steps (a) to (e). In this final cycle, step (e) is optional.

[0207] Furthermore, at least one cycle of step (b) or (d) includes applying tension to one or more template strands that tend to stretch the nucleic acid. In some versions, the process may be carried out at a constant temperature.

[0208] According to embodiments of the present invention, precise control may be achieved using hydrodynamic focusing, such as that taught in Wong et al., "Deformation of DNA molecules by hydrodynamic focusing," J. Fluid Mech., 2003, vol. 497, pp. 55-65, Cambridge University Press; and / or using mechanical force, such as that taught in Liphardt et al., "Reversible Unfolding of Single RNA Molecules by Mechanical Force," Science, 2001, vol. 292, pp. 733-737, American Society for the Advancement of Science; the teachings of both references are incorporated herein by reference in their entirety. Other techniques may be used.

[0209] The level of precision control can be on the nanometer scale, for example, if DNA may have a diameter of 2 nm and protein may have a diameter of 10 nm. According to embodiments of the present invention, control of the translational position or rotational orientation of molecules can be achieved within less than 100 nm, less than 10 nm, or less than 1 nm. Furthermore, the reaction chamber itself can be small in size, with the largest dimensions being less than 10 mm, less than 1 mm, less than 100 microns, less than 10 microns, less than 1 micron, less than 100 nm, less than 10 nm, or less than 1 nm. In addition, temperature, pressure, diffusivity, tension, chemical concentration, salt concentration, enzyme concentration, or other parameters that regulate or control the reaction can vary within a tightly controlled range, for example, plus or minus 10%, plus or minus 1%, plus or minus 0.1%, plus or minus 0.01%, plus or minus 0.001%, or plus or minus 0.0001%.

[0210] Where used herein, if a system is “configured” to perform precise control of parameters, such control of parameters may be performed, for example, by using one or more software or hardware modules that are controlled under the control of a computer processor in a mobile device according to embodiments of the present invention. The computer processor may include one or more control modules that are specially programmed to receive digitally coded signals corresponding to the values ​​of one or more parameters to be controlled and to perform closed-loop control of these parameters to be controlled. For example, a control module may receive the values ​​of a parameter and, based on the values ​​of the parameter, adjust one or more forces or other conditions applied to a molecule being analyzed (e.g., by using the application of tension). As a result of the adjustments performed by the control module, the parameter changes, and a closed feedback loop may be formed, under which the control module performs closed-loop control of one or more parameters to be controlled. In addition to receiving digitally coded signals from one or more other computer modules, a control module may receive one or more sensor inputs from one or more sensors that sense the values ​​of the parameters to be controlled; for example, a temperature or pressure sensor may provide a temperature or pressure sensor input to the control module.

[0211] Hardware system As described above, the compact integrated chips having biological assays are intended to be disposable. The present invention also includes hardware systems comprising mechanical, electrical, and electronic devices and computer-readable instructions for controlling these devices. Those skilled in the art will understand that such hardware systems can be configured to house chips of various designs utilizing the above-mentioned biological processing modules (e.g., nucleic acid extraction, thermal or isothermal amplification, laser-based fluorescence detection, and CE separation).

[0212] Figure 11 is a block diagram of the hardware system 400 included in the present invention. The details of the configuration of the hardware system 400 will be further described below with reference to Figure 14.

[0213] Referring to Figure 11, the chip 20 (which may be disposable) is typically inserted into a hardware module 400 controlled by a microprocessor / computer interface 408. As shown in Figure 11, the hardware module 400 includes an extraction control module 402, an amplification control module 404, and a detection control module 406. The centrally controlled hardware module 400 may be operated to adapt to sequential processing on the chip 20, or modules 402, 404, and 406 may each be controlled to perform operations on any one or a combination of the modules. The characteristics of each hardware module 400 are as follows:

[0214] The microprocessor / computer interface 408 means may include, for example, a suitable computer such as any of those taught herein, and conventional computer elements such as any of those taught herein; and may include RAM for storing an operating system such as any of those taught herein, and software suitable for signal processing related to the detection of nucleic acids.

[0215] Extraction control module Referring again to Figure 11, a magnet or electromagnet of 12,000–13,000 gauss is added to the extraction module 402, either below or above the extraction chamber (chamber 104 shown in Figure 4), as needed. The application of the magnetic field is controlled for periods ranging from just one second to several tens of minutes, allowing for the aggregation and retention of the magnetic beads as desired by the protocol. If desired, DNA can be eluted from the beads using heat, as described in the extraction protocol. Thus, in addition to the application of the magnetic field, the application of heat of approximately 50–90°C for any appropriate length of time during amplification can be achieved by the hardware system 400 using the same thermal module used in the amplification module 704, which will be described next.

[0216] Amplification control module In Figure 11, the temperature of the amplification module 404 cycles between approximately 40 and 95°C with respect to thermal cycling. This is achieved using a Peltier device or any other suitable heating / cooling device that can be used for amplification. With respect to the desired time interval and repeating cycle, up to three thermal cycle temperatures allow for the processing of DNA denaturation (70–100°C), primer annealing (approximately 40–80°C), and extension temperature (50–90°C), thereby producing exponential DNA amplification. The designated amplification chamber on the chip 20 is placed on top of a temperature-cyclable Peltier device.

[0217] Figure 12 shows a block diagram of an exemplary embodiment of the Peltier device 550 used in the amplification control module 404 (Figure 11). The computer interface 408 controls a microprocessor 556 that performs digital proportional / integral / derivative (P / I / D) feedback control to the thermal control unit 562. The digital-to-analog (D / A) converter 558 translates digital values ​​from the microprocessor 556 into analog voltages or currents applied to a power supply 560 connected to the thermal cycle unit 562. Adjusting the analog voltage or current changes the output of the power supply 560, which in turn changes the temperature of the thermal cycle unit 562. Since a chip (not shown) is connected to the thermal cycle unit 562, temperature changes in the thermal cycle unit 562 heat or cool the chip accordingly. A temperature sensor 554 monitors the temperature of the chip and / or the thermal cycle unit 562 and provides analog signal parameters, such as analog voltages, to the analog-to-digital (A / D) converter 552. The A / D converter 552 translates analog signal parameters into digital signals suitable for use by the microprocessor 556.

[0218] Detection and control module Figure 13 is a block diagram of a detection control module 406 according to the system of the present invention. Laser-guided fluorescence combined with high-voltage electrophoresis is used for the detection of amplified DNA, or, in some embodiments, isolated proteins. The detection control module 406 uses a programmable high-voltage source 620 to supply the required voltage signal (400V to 10kV) via electrodes 622 along a designated channel on the chip 20. A safety feature is incorporated that shuts down the high-voltage source 620 if the enclosure (not shown) housing the electronics and chip is not closed and stable.

[0219] A laser source 602 having a desired wavelength and a suitable filter configuration (not shown) irradiates a selected portion of the tip 20 through lens 606. The irradiation excites fluorophores bound to DNA or proteins, causing the fluorophores to emit radiation (not shown) at a different wavelength from the laser wave. The filter blocks the laser light and transmits the emitted radiation, which is collected by lens 607. Lens 607 focuses the filtered radiation onto a detector 608, such as a photomultiplier tube, avalanche light-emitting diode, charge-coupled device, or other suitable detector. The detector emits a signal, such as a photocurrent, amplified (and filtered as necessary) by a signal amplification circuit 610. Computer 408 records and processes the amplified signal; and can also control the high-voltage source 620 and laser 602 via a software interface using computer 408, as follows. Those skilled in the art will understand that fluorescent markers bound to DNA can be examined using alternative configurations of optical elements.

[0220] Detailed structure of hardware system 400 Figure 14 is a block diagram of a hardware system 400 according to an embodiment of the present invention. The holder 752 houses a compact integrated chip (not shown), such as the chip 20 in Figure 4, in a groove 758. The chip can be secured within the holder 752 using an adjustable clamp or any other suitable support means. The heater 562 (Peltier assembly 562 in Figure 12) and magnet 756 are positioned below the holder 752 so as to be adjacent to the appropriate portion of the chip held by the holder 752. In a preferred embodiment, the heater 562 and magnet 756 can be moved to accommodate chips of different sizes or configurations.

[0221] The cover 754 may be positioned on top of the chip, which is held in the groove 758 of the holder 752, by a hinge or other suitable mechanism. By positioning the cover 754 on top of the chip, the electrodes 776 are inserted into the appropriate wells of the chip (e.g., CE wells 114, 116, 118, and 120 of the chip 20 in Figure 4). Connected to a high-voltage source 620 controlled by the computer 408, the electrodes 776 can apply electric fields of different intensities (e.g., 400 to 6000 V) across the entire area of ​​the corresponding portion of the chip, such as the CE channel 112 shown in Figures 4 and 8A to 8C.

[0222] The hardware system 400 also includes a detection system similar to that shown in Figure 13. Laser 602 produces a laser beam 760 (solid line) reflected by a beam splitter 766 and two mirrors 781 and 783, illuminating a portion of the chip containing fluorescently tagged nucleic acids, such as the detection channel 112 shown in Figures 4 and 8A–8C. When irradiated by laser beam 760, the fluorescent tag emits fluorescent radiation. A portion of this fluorescent radiation forms a fluorescent beam 762 (dotted line) that propagates along the path of laser beam 760, but in the opposite direction. A portion of the fluorescent beam 762 propagates through the beam splitter 766 and through a filter 764 that filters out light of wavelengths different from the fluorescence wavelength. The filtered fluorescent beam 762 irradiates a detector 608 (e.g., a photomultiplier tube, avalanche light-emitting diode, charge-coupled device, linear array, or other suitable detector) which transmits a corresponding signal, such as a photocurrent, to an amplifier 778. The amplifier 778 amplifies the signal from the detector 608 and transmits it to the computer 408, which can record, process, and display the results related to the fluorescence measurement.

[0223] Software Interface Some embodiments of the present invention include a software module configured to control three biological hardware modules, each using a graphical user interface (GUI) (i.e., the extraction control module 402, amplification control module 404, and detection control module 406 of the aforementioned hardware system 400, as shown in Figure 11).

[0224] The GUI allows the user to control the extraction module through user input or a pre-programmed magnetic field initiation. In some embodiments, the magnetic field may be applied discontinuously for specified time intervals, as instructed by the protocol described above. The GUI also allows the user to specify temperature application for a specified time period.

[0225] The GUI allows the user to set user-specified temperatures (typically three different temperatures as described above) and the time interval for each temperature. The user can also specify a desired number of thermal cycles by repeating the above input temperature and time parameters. The GUI allows the user to configure microprocessor / computer data acquisition of chip / sample temperature from the hardware setup; the resulting data can be displayed in real-time as a graph comparing temperature against time.

[0226] Furthermore, the GUI provides the user with the ability to (1) select and power any pair of electrodes to be configured for the application of a high-voltage signal; (2) sequentially specify the above selection over a given time interval as desired by the user; (3) obtain feedback monitoring the voltage applied across the entire area of ​​any of the powered electrode pairs and graph this data as a function of time; (4) obtain feedback monitoring the current of any of the powered electrode pairs and graph this data as a function of time; (5) graph the acquired fluorescence signal as a function of time; and (6) identify / distinguish the fluorescence signal (peak / intensity) from a size standard signal. Using these features, amplification can be confirmed and the presence (or absence) of a sequence in the DNA can be identified.

[0227] The hardware system and computer-readable instructions of the present invention also facilitate the comparison of data obtained by analyzing biological samples using the integrated chip described herein with a genome database storing multiple genome profiles.

[0228] Microfluidic valve Microfluidic valves and pumps are typically used within a chip, such as the chip 20 in Figure 5 (which may be the chip 20 in Figure 4 in one embodiment), to achieve controlled flow and to retain fluids and vapors within the chip.

[0229] For example, referring to Figure 4, a rubber plug may be used as a valve in some embodiments of the present invention. Such a rubber plug is inserted, for example, into the sample input well 100 and reagent inlet / outlet port 106 of the tip 20 during nucleic acid extraction to seal the extraction chamber 104. Similarly, during amplification, the amplification chamber 108 may be sealed by inserting a rubber plug into the reagent inlet / outlet port 106 and sample well 120.

[0230] In other embodiments, various valve systems may be used. Such systems are described elsewhere in this specification, see, for example, Figures 5, 6A–6B and 7A–7B.

[0231] Another embodiment of the integrated microfluidic chip involves a valve technology called an “elastic valve,” an example of which is shown in Figures 15A and 15B. As shown in Figures 15A and 15B, the elastic valve 1100 uses an inflatable elastic encapsulating membrane 1110, similar to a catheter used in medical surgery. The fluid path includes a channel 1102 formed between an upper chip substrate 1104 and a lower substrate 1106. The inflation or operation of the membrane 1110 can be achieved by an inlet air tube 1112 connected to the membrane 1110, as shown in Figures 15A and 15B (i.e., the membrane 1110 is actuated and controlled by air pressure). When the deflated membrane 1110 is positioned at an open port 1108, as shown in Figure 15A, a fluid flow 1114 across the channel 1102 is possible. However, as shown in Figure 15B, when the elastic valve is inflated by air 1116, it seals the expanded and open port 1108, and thus restricts the fluid flow 1114 across the open port 1108 of channel 1102. In an integrated chip, a series of such valves 1100 can be used to achieve flow control as needed.

[0232] In alternative configurations, the elastic valve can be embedded within the chip rather than operated via an insert or external interface, achieving a higher level of automation. An example of such a configuration would involve a fabrication process in which the elastic membrane is sandwiched between two layers of the chip along any channel requiring fluid control. The inlet air tube used to actuate the elastic membrane valve is also embedded within the chip, and brought to the outer periphery of the chip for connection to an external pneumatic line. Both embedded and external valve and pump systems can be effectively used to regulate volumes ranging from nanoliters to several milliliters using the valve / pump.

[0233] Figures 16A–16F show an elastic valve assembly 1200, which includes a series of elastic valves, such as the elastic valve 1100 shown in Figures 15A and 15B, and is configured to enable pump operation similar to that of a parasitic pump. The elastic valve assembly 1200 includes an open well 1202a into which fluid is loaded and connected via channel 1206a to a closed chamber 1208. The closed chamber 1208 is connected via a second channel 1206b to a second open well 1202b. Elastic membranes 1204a and 1204b control the fluid flow in channels 1206a and 1206b, respectively, according to the principle described above. A third elastic membrane 1204c, located in the closed chamber 1208, controls whether fluid enters or leaves the closed chamber 1208. The pressure inlet tubes 1212a to 1212c, which are connected to the elastic membranes 1204a to 1204c respectively, allow the membranes 1204a to 1204c to expand and contract.

[0234] As shown in FIGS. 16A-16F, by sequentially expanding and contracting three membranes 1204a-1204c, fluid can be drawn from an open well 1202a to a closed chamber 1208. First, as shown in FIG. 16A, load fluid into the open well 1202a. Then expand the elastic membranes 1204a-1204c sequentially as shown in FIGS. 16B-16D. After all three membranes 1204a-1204c are expanded, contract the membrane 1204a of the channel 1206a to create a suction force that draws fluid from the open well 1202a to the channel 1206a. Contracting the membrane 1204c in the closed chamber 1208 creates an additional suction force that draws fluid from the channel 1206a into the closed chamber 1208. The fluid can also be unloaded from the closed chamber 1208 to either the open wells 1202a and 1202b by sequential operation of the similar membranes 1204a-c.

[0235] System and method of using an integrated chip In embodiments according to the present invention, there is provided a portable system for extracting, optionally amplifying, and detecting nucleic acids, using a compact integrated chip in combination with a portable system that analyzes a detection signal and compares and provides the results via a wireless network. A portable chip-based diagnostic device can be used for rapid and accurate detection of DNA / RNA signatures of a biological sample. The portable device can be used as a platform for personalized and passive nanomedicine or companion diagnostics, or as a tool to help improve efficiency, reduce toxicity, and expedite clinical trials and regulatory approval of new drugs.

[0236] In one embodiment, the system can be used in a method of practicing personalized medicine. Broadly speaking, in personalized medicine, genetics is used to provide the ideal drug at the ideal dosage for the ideal patient for an ideal outcome. In embodiments according to the present invention, a portable assay system is used to extract, amplify, and detect nucleic acids of a sample, and in particular, to detect nucleic acid-based personalized biomarkers. The system can then determine a dosage and / or combination of drugs suitable for the delivery of a customized medicine based on the detected personalized biomarkers. It can provide a target drug and a companion diagnosis. Further, the system can be used to determine whether a person will respond to drug treatment. Also, the system can be used to facilitate patient stratification, enhance drug safety and efficiency, and facilitate optimization of dosing and treatment regimens. Further, the system can be used for human monitoring, for example, by monitoring the amount of nucleic acids found in a person's biological samples taken at different times. Such monitoring can be used, for example, to follow the progress of a patient's treatment or to monitor a person's disease. For example, diabetes and other chronic diseases can be diagnosed, classified, or monitored via DNA / RNA markers such as inflammatory markers. For example, determining a personalized genomic profile can include detecting nucleic acids indicative of the type or subtype of diabetes.

[0237] In another embodiment, using a portable system according to an embodiment of the present invention to enrich an experimental group can help reduce the size and cost of a regulated clinical trial. Individualizing the trial with a subset of genes can dramatically enhance the treatment effect and shorten the approval process. The resulting combination of drug and companion diagnosis has additional value for the target group of genes.

[0238] In another embodiment, a portable system according to the present invention may be used to provide personalized care, for example, in the fields of cosmetics, medicinal cosmetics, and skin care applications. For example, a portable assay system may be used to extract, amplify, and detect nucleic acids from a sample; in particular, it may be possible to detect personalized biomarkers based on nucleic acids. The system may then determine the type, quantity, or combination of personalized care products to deliver based on the personalized biomarkers. For example, the system may be used for the selection and delivery of cosmetics based on personalized cosmetic biomarkers. In one example, skin type may be determined based on personalized biomarkers, and then this may be used to determine the type, quantity, or combination of cosmetic products to deliver to a person. The presence of key biomarkers (which may be DNA or RNA-based, for example) may be measured and quantified in real time using a portable device. Sequence biomarkers (e.g., cosmetic biomarkers such as age-related loci, specific aging genes or gene expression patterns, and skin quality) may be measured against skin products. An individual's genotype may be correlated with such sequence biomarkers using a portable system according to embodiments of the present invention. An integrated chip may be customized to conform to a library of target genes for cosmetic biometrics. Individual beauty biometrics can be measured by quantifying them in real time via a portable system according to an embodiment of the present invention. These biomarkers can then be observed to see how they change over time, along with the use of corresponding beauty products. The embodiments may also be used for wellness applications, for nutrigenomics, and for Ayurvedic genomics, which performs Ayurvedic diagnostics via genes corresponding to body types such as Vata, Kapha, and Pitta.

[0239] In another embodiment, a portable system according to the present invention may be used, in which the detection capability of the integrated chip is integrated with a specific, independently determined pharmaceutical product. For example, the portable assay system may be used to extract, amplify, and detect nucleic acids from a sample; in particular, it may be used to detect personalized biomarkers based on nucleic acids, where the personalized biomarker may indicate the presence of a specific strain of disease or pathogen. The system may then independently determine a customized dosage and / or combination of drugs to deliver based on the biomarker of the specific strain. In one example, the dosage and / or combination of drugs to deliver may be determined with respect to a specific strain of human immunodeficiency virus (HIV). The portable system according to the present invention may be used as a drug monitoring device or tool for point-of-care detection of HIV strains, determination of HIV viral load and HIV genotyping, or for monitoring and preventing mother-to-child transmission of HIV.

[0240] More generally, embodiments of the present invention can be used to identify the genotype of any organism, thereby determining at least one of the following: predisposition to genetic disease, disease strain, and antibiotic resistance to disease conditions. For example, the type of viral hepatitis can be determined; or oncogenes can be identified.

[0241] In various embodiments, portable systems according to the present invention can be used in a variety of different potential industries. For example, the portable assay system can be used to extract, amplify, and detect nucleic acids from a sample. The detected nucleic acids can then be used in any of the various different potential industries (in addition to those discussed elsewhere in this specification). For example, the detected nucleic acids may be related to food safety, agricultural diseases, veterinary applications, archaeology, forensic science, nutritional methods, nutritional supplements, nutrigenomics, water testing / satellite, food and beverages, environmental monitoring, tariffs, safety, defense, biofuels, sports and wellness; and they may also be used for theragnosis.

[0242] Using a system according to embodiments of the present invention, a personalized genomic profile of a person who is the source of a biological sample can be determined. The personalized genomic profile can then be used to personalize nutritional supplements, medicinal cosmetics, pharmaceuticals, foods or beverages, or nutritional regimens for that person. The personalized genomic profile can be used, for example, for personalized wellness, personalized sports nutrition, personalized meals, or personalized nutritional regimens. In the field of sports, for example, inflammatory markers can be determined to detect, measure, and treat injuries in athletes, such as traumatic brain injury in contact sports. In another embodiment, markers can be determined to assess blood doping in sports. In another embodiment, various types of phenotypic expression of RNA markers can be measured.

[0243] A system according to an embodiment of the present invention may be used for personalized nutrition, in which a chip reader (such as a nucleic acid extraction control module, a nucleic acid amplification control module, and a nucleic acid detection control module) and software are customized to help personalize food and beverages and nutrition based on a person's DNA / RNA profile. In a broad sense, personalized nutrition includes using genetics to provide the ideal nutrition for ideal results to the ideal consumer in real time. In embodiments according to the present invention, personalized nutritional supplements may be provided to a person based on their DNA / RNA profile. Targeted nutrition and companion diagnostics may be provided, and companion diagnostics for health and wellness biomarkers may be developed. A holistic and integrated approach to health and wellness may be provided. Consumers may be provided with feedback measuring progress not only by generally accepted phenotypic markers but also by DNA / RNA biomarkers that provide robust and reliable progress measurements. A person's diet may be personalized by determining predispositions based on their genetic profile or DNA / RNA markers. Using a personalized genomic profile, a person's nutritional predispositions, such as genetic food allergies, intolerances, or sensitivities, including gluten sensitivity, gluten intolerance, or lactose intolerance, can be determined, and diets can be personalized based on that profile. Inflammatory markers can be measured as a method to monitor whether or not a person is tolerant to a particular diet. Furthermore, nutritional deficiencies can be detected, measured, and then addressed by adding micronutrients to food or beverages. For example, vitamin deficiencies, mineral deficiencies, and other deficiencies (such as vitamin B12 deficiency, folic acid deficiency, or iron deficiency) can be measured based on biomarkers measured by embodiments according to the present invention. The deficiencies can then be addressed by providing appropriate micronutrient supplements to food or beverages. Furthermore, probiotics or prebiotics that are deficient or altered in a person's microbiome can be determined.For example, a biological sample from a person's saliva, gastric juice, or urine can be obtained and analyzed to determine which probiotics or prebiotics are deficient or altered in that person's microbiome, such as the microbiome of their mouth or stomach. The deficient probiotics or prebiotics can then be supplemented in the person's food or beverages to help restore their normal microbiome.

[0244] In embodiments of the present invention, personalized nutrition can be implemented based on the determination of inflammatory biomarkers. Inflammation is associated with a variety of conditions, including cancer, cardiovascular conditions, Alzheimer's disease, lung disease, arthritis, autoimmune diseases, neurological disorders, and diabetes. In embodiments of the present invention, a person's diet can be personalized by determining their genetic profile or predisposition based on DNA / RNA markers. As an example, selected cardiovascular inflammatory biomarkers that can be determined, measured and / or monitored according to embodiments of the present invention are listed in Figure 27. Selected diabetic inflammatory biomarkers that can be used are listed in Figure 28. In personalized medicine and personalized nutrition, the biomarkers shown in Figure 29 may be used, for example, to determine the progression of obesity, diabetes, and cardiovascular disease. Other biomarkers other than those shown in Figures 27-29 may also be used, and it is understood that such biomarkers may be used for personalized nutrition, personalized medicine, and other techniques described herein.

[0245] In another embodiment according to the present invention, the consumer experience of food or beverage can be enhanced by distributing food or beverage based on a person's nutritional genomic profile. The system taught herein can be embedded in the shape factor of a distribution activator that can be controlled based on control inputs from the system taught herein, such as at least part of a vending machine, automated counter, or kiosk. For example, a person may provide a biological sample to a gene analysis unit configured to determine biomarkers associated with that person. Based on the biomarkers, an individualized genomic profile of the person from whom at least one biological sample is derived can be determined. A distribution control unit is then configured to determine the product or portion of a product to distribute based on the individualized genomic profile. A distribution activator is connected to the distribution control unit and configured to distribute at least part of the product under the control of the distribution control unit. Using such a system, for example, a consumer may insert a biological sample, such as a saliva sample, into a vending machine that can determine which food or beverage is most appropriate for the consumer based on the consumer's nutritional genomic profile. The dispensing activator may comprise a 3D printer configured to print at least a portion of a product, or the dispensing activator may be configured to dispense a pre-packaged product. For example, the product may be food, beverage, medicinal cosmetics, nutritional supplements, pharmaceuticals, herbal medicines, alternative medicines, and cosmetics. The dispensing control unit is configured to determine the product or portion of a product to dispense based on nutritional predispositions, nutritional deficiencies, and at least one probiotic or prebiotic that is deficient or altered in the microbiome, any of which may also be determined by detecting nucleic acids based on embodiments taught herein. For example, a 3D printer may be used to print a consumer's food or beverage in a vending machine or kiosk based on the consumer's genetic profile. The consumer may input additional information using a user interface such as a keyboard, touchpad, speech recognition interface, augmented reality interface, or other user interface.Consumers may be given some information about alternative ingredients that have benefits based on their profile, and then, using a user interface, select one or more of these ingredients to order and have food or beverages made. Systems such as vending machines may integrate, for example, at-time companion diagnostics and at-time production of nutritionally fortified food or beverages using 3D printing of food or beverages. Food, beverages, medicinal cosmetics, nutritional supplements, and cosmetics can be printed in 3D in this manner. In the case of cosmetics, for example, a customer may be provided with a customized profile using the system taught herein at a kiosk cosmetics counter, and then have customer-specific cosmetics, such as skin care solutions, printed. Similar systems may also be provided for nutritional supplements, herbs, alternative medicines, herbal teas, food products, and beverages. For example, spices or other ingredients such as turmeric, cinnamon, green tea, or other anti-inflammatory ingredients may be added to juices to reduce a consumer's inflammation levels. Specially tailored dosages may be provided by a control system based on the customer's genetic profile in the following embodiments.

[0246] In another embodiment according to the present invention, the system taught herein is used in conjunction with an enhanced extracorporeal counterpulsation (EECP) therapy machine. The amount of cardiac markers, inflammatory markers, and other markers associated with improved cardiac function is monitored in real time by the nucleic acid analysis system taught herein and used to provide electronic control inputs to the EECP machine, for example, to increase or decrease blood flow or titration. For example, inflammatory factors, VEGF, endothelial growth factor, other gene expression patterns, cardiac markers, DNA / RNA markers correlated with cardiac status, etc., can be monitored by the system taught herein. By linking the nucleic acid analysis system taught herein with the EECP therapy machine, a tool can be provided that helps reverse atherosclerosis with a combined effect produced by stimulating endothelial repair through the measurement of flow and inflammatory factors. Biomarkers related to the atherosclerosis process can be monitored simultaneously on multiple channels of the nucleic acid analysis system, and based on the amount of biomarkers, electronic control inputs can be provided to the EECP machine.

[0247] In various embodiments, the results provided by a portable system according to the present invention include the amount of virus loaded (e.g., number of copies per ml), the predicted cell count per volume (e.g., mm²), and 3 This may include predicting CD4 counts per cell and titrating drug delivery.

[0248] In further embodiments of the present invention, the portable system according to the present invention may communicate with other systems in a variety of different possible ways. The portable system may transmit and receive regulated data signals related to biological samples and may transmit information via wired media (e.g., wired network or wired direct connection) or wireless media (e.g., acoustic, infrared, radiation, microwave, spread spectrum). The portable system may transmit information via, for example, the World Wide Web and / or mobile networks and / or text messages (such as SMS messages). The portable system may connect to a remote genome database storing genome profiles. The portable system may store, transmit, or receive signal profiles of a single reference sample.

[0249] In further embodiments of the present invention, a portable system or mobile device may be implemented as part of any of a wide variety of possible handheld or tablet devices, such as a smartphone, personal data device (PDA), mobile phone, or other handheld or tablet device, utilizing a compact integrated chip that may be disposable, or may interface with any of a wide variety of possible handheld or tablet devices, such as a smartphone, personal data device (PDA), mobile phone, or other handheld or tablet device. Furthermore, similar graphical user interfaces and external designs may be used for use on such widely available handheld or tablet devices. In one embodiment, all embodiments of the present invention may be implemented with, or interface with, or use a graphical user interface or external interface similar to those of the iPhone, iPad, or iPod sold by Apple Inc. in Cupertino, California, USA, or the Galaxy sold by Samsung Electronics Co., Ltd. in Suwon, South Korea.

[0250] In embodiments according to the present invention, disease outbreaks can be tracked in widely dispersed analysis sites using a portable system or multiple such portable systems in distributed locations. The portable systems may be connected via one or more networks (e.g., local area networks, wide area networks, and / or the Internet) and may engage in bidirectional information exchange between a central data center and system end-users. The data center may provide the end-user / inventive system with known pathogen / disease mapping information with respect to biological sample analysis, and the inventive system / end-user may subsequently transmit assay results to the data center. The data center may receive geographic location information and other case-specific information from the end-user / inventive system. The data center may monitor assay results coming from multiple deployed units / inventive systems and, using pattern detection programs, track disease outbreaks, for example. When the data center detects a threshold pattern, it programmatically generates notifications to remote portable systems according to the present invention.

[0251] Further use and application of systems and integrated chips In various embodiments, the system of the present invention can be used to identify pathogens, diagnose diseases with genetic markers, or identify an individual's genotype. The method of the present invention typically includes (1) providing at least one integrated chip; (2) loading at least one biological sample onto at least one integrated chip; (3) functionally linking a portable control assembly with at least one integrated chip; and (4) activating the portable control assembly to perform extraction, amplification, and detection of nucleic acids from the biological sample loaded onto the integrated chip.

[0252] The present invention can be used to diagnose and detect a wide range of pathogens and disorders having nucleic acid-based genetic materials and / or genetic components. In addition to detecting targets such as HIV, HBV, HCV, and sexually transmitted infections, the system and methods of the present invention can be used to detect and diagnose molecular diagnostic targets arising in the fields of oncology, cardiovascular medicine, specific testing, and prenatal screening.

[0253] Preferably, the biological sample is derived from a biological fluid, such as blood, saliva, semen, urine, amniotic fluid, cerebrospinal fluid, synovial fluid, vitreous fluid, gastric juice, nasopharyngeal aspirate, and / or lymph.

[0254] The biological sample may be a tissue sample, water sample, air sample, food sample, or crop sample. Preferably, the biological sample analysis detects one or more of the following: waterborne pathogens, airborne pathogens, foodborne pathogens, or cropborne pathogens.

[0255] The pathogens detectable by the system and method of the present invention may originate from a variety of hosts. Whether living or non-living, the host should be capable of supporting the replication of infectious pathogens by enabling them to replicate within or on the host. Examples of such hosts include liquid or solid in vitro culture media, animal cells or tissues, plants or single-celled organisms, and all organisms encompassing mammals such as humans.

[0256] The systems and methods of the present invention may be used in one of many of the following areas. In one embodiment, the systems and methods of the present invention may be used in the field of defense against biological weapons. For example, the present invention may be used for time-of-occurrence and real-time pathogen detection. In another embodiment, the systems and methods of the present invention may be used in the field of life sciences. For example, the present invention may be used as a portable analytical instrument and in conjunction with a portable analytical instrument. In another embodiment, the systems and methods of the present invention may be used in the field of clinical diagnosis. For example, the present invention may be used to diagnose and / or identify pathogens by doctors, nurses, or untrained users in hospitals, homes, or the field. The present invention may also be used to identify the genotype of an organism, thereby determining predisposition to genetic diseases, if any, or predisposition to antibiotic resistance, if any. The present invention may also be used to determine the pathogens present in a patient and the susceptibility and resistance profiles of those pathogens to various antibiotics. The present invention may also be used as a drug monitoring device, a disease prognostic indicator, and a diagnostic and therapeutic device. In another embodiment, the systems and methods of the present invention may be used in the field of industrial and agricultural monitoring. For example, the present invention can be used to monitor and / or detect pathogens transmitted by food, crops, livestock, etc. In another embodiment, the systems and methods of the present invention can be used in the field of forensic medicine. For example, the present invention can be used to genetically identify individuals.

[0257] In one embodiment, genetic disorders and disorders containing genetic components can be diagnosed using the systems and methods of the present invention. For example, many oncogenes have been identified, including p53, which is involved in the progression of breast cancer, colorectal cancer, and other cancers; c-erbB2, which is involved in the progression and metastasis of breast cancer; and BRCA1, which is involved in 50% of all hereditary breast cancers and is also associated with an increased risk of prostate cancer and other cancers. Screening for these genetic markers can be achieved using the systems and methods described herein.

[0258] Infectious pathogens that can be diagnosed using the system and method of the present invention include, but are not limited to, bacteria, viruses, fungi, actinomycetes, and parasites.Examples include, but are not limited to, Escherichia, Shigella, Salmonella, Arizona (Salmonella subgenus III), Citrobacter, Klebsiella, Enterogacter, Serratia, Proteus, Providentia, Morganella; Vibrio and Campylobacter; Brucella such as Brucella abortus; Yersinia; Pasteurella; Francisella; Actinocacillosis; Haemophilus, Bordetella (e.g., Bordetella pertussis); Pseudomonas and Legionella; Bacteroides, Fusobacterium, Streptobacillus and Calymmatobacterium; Bacillus (spore-forming aerobic bacteria); Clostridium (spore-forming anaerobic bacteria); Lissteria and Erysipelothrix; Corynebacterium; Mycobacterium; Spirochetes; Rickettsias; Chlamydia; Mycoplasma; Poxviruses; Herpesviruses; Papovaviruses; Adenoviruses; Orthomyxoviruses, Paramyxoviruses; Rhabdoviruses; Cytomegaloviruses; Retroviruses; Picornaviruses; Coronaviruses; Rotaviruses; Hepatitis viruses (e.g., Hepatitis C virus, Hepatitis B virus); Togaviruses; Bunyaviruses; Arenaviruses; Cryptococcus; Candida (e.g., Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis); Sporothrux; Ilestoplasma; Coccidioides; Blastomyces; Aspergilli; Zygomycetes; Dematiaceae; Fusarium; Protozoa; Nemathelminthes; and Platyhelminthes.

[0259] In other embodiments, retroviruses such as HIV-1, HIV-2, any of HIV-1 group M, N, O, or P, any of HIV-1 group M subtypes A to K, or other known types or subtypes of HIV, HTLV-1, and HTLV-2, as well as herpesviruses such as HSV-1, HSV-2, VZV, EBV, CMV, and HHV-6, can be detected using the system and method of the present invention.

[0260] In other embodiments, disorders and diseases that can be detected using the systems and methods of the present invention include: Borellia, human T-cell lymphoma / leukemia virus-I, human T-cell II / leukemia virus-II, human immunodeficiency virus, influenza virus, parainfluenza virus, adenovirus, HSV, VZV, HPV (genus 6, 11, 16, 18, 31, 33), Moraxella catarrhalis, Streptococcus pneumonia, Haemophilus influenzae, Legionella spp., human parvovirus B19, and Group B streptococcus.

[0261] In other embodiments, disorders and diseases that can be detected using the systems and methods of the present invention include genetically predisposed genetic disorders and disorders. Examples, but not limited to, include cystic fibrosis, Gaucher disease, medium-chain acyl-CoA dehydrogenase deficiency, myotonic dystrophy, sodium channel disease, chlorine channel disease, and Duchenne / Becker muscular dystrophy.

[0262] In certain embodiments, the following pathogens may be diagnosed: human immunodeficiency virus, TB bacteria, hepatitis C virus, SARS virus, Chlamydia trachomatis, cytomegalovirus, Gardnerella vaginalis, Group A Streptococcus, Group B Streptococcus, human papillomavirus, Neisseria gonorrhoeae, Trichomonas vaginalis, Bordetella pertussis, and E. coli.

[0263] In one embodiment, the infectious pathogen detected is methicillin-resistant S. aureus (MRSA). Staphylococcus aureus is one of the most prominent pathogens causing hospital-acquired and community-acquired infections. Beta-lactam antibiotics are preferred agents for serious S. aureus infections. Since the introduction of methicillin into clinical practice in 1961, methicillin-resistant S. aureus (MRSA) strains have steadily increased, making hospital-acquired infections a serious global problem. Therefore, the detection of methicillin resistance is of crucial importance for the treatment and management of patients. In clinical laboratories, S. aureus is identified by detecting growth characteristics and subsequent catalase and coagulase activity or specific surface components. DNA-degrading enzyme and thermostable endonuclease tests are used as confirmatory tests for indeterminate or negative coagulase tests. Conventional susceptibility testing for S. aureus reliably detects resistance to methicillin or oxacillin when agar dilution or agar screening methods are used according to NCCLS standards. Methicillin resistance is associated with the production of penicillin-binding protein encoded by the mecA gene, or, in rare cases, with the overproduction of beta-lactamase.

[0264] Conventional methods for detecting MRSA utilize broth and agar-based antimicrobial susceptibility tests to provide a genotypic profile of a given microorganism's response to a range of pathogens. These methods are time-consuming, problematic, and often produce false positives and false negatives. These shortcomings stem from the expression of the staphylococci resistance gene, which can be expressed in a highly heterogeneous manner, making it difficult to characterize the resistance genotype. Molecular methods targeting the detection of the resistance gene mec A can be employed. Screening for mutations in the amplified product can be facilitated by the use of high-density probe arrays such as Affimetrix® Genechips®, which may be used in the implementation of this invention.

[0265] Similarly, detecting drug resistance by identifying specific antibiotic resistance genes (identification of resistance genotypes) can be achieved in many organisms. Examples are listed in Table 1 below: [Table 1]

[0266] In other embodiments, the viral and microbial diseases that can be detected using the system and method of the present invention are listed in Table 2: [Table 2]

[0267] In further embodiments, the infectious diseases listed in Table 3 can be tested using the system and method of the present invention: [Table 3]

[0268] Use across multiple sectors and industries Based on the above features and effects of the present invention, many developments are possible in healthcare (and other industries), particularly across multiple sectors. These sectors in the healthcare industry include:

[0269] government departments (e.g. Centers for Disease Control, military operations, Department of Agriculture, etc.);

[0270] Commercial sector (e.g., hospitals / medical institutions, analytical / diagnostic laboratories, pharmaceutical companies, etc.),

[0271] This includes the private sector (for example, doctors, farmers / large-scale farm owners, and individuals).

[0272] In exemplary embodiments, a laboratory or lumber company serves as a supply point for the various prepared aggregate chips described above. Different chips are prepared to analyze different pathogens and diseases. End users of the chips are from various sectors and deploy portable systems in their respective locations and environments, including patient use at home, field use (e.g., crop and veterinary checkpoints), remote / regional locations, and hospitals / medical centers, customs ports, etc. Due to the plug-and-play configuration between the aggregate chips and portable systems of the present invention (i.e., rapid reader response time, chip assay versatility, and chip disposability), a given portable assay system / device of the present invention can load different chips (to detect different pathogens / diseases) at a given location, or it can be limited to loading one type of chip (to detect a specific pathogen / disease).

[0273] When using a portable assay system, a bidirectional exchange of information occurs between a central data center (where the operational data library and bioinformatics data reside or are maintained) and the system end-user. In this bidirectional exchange, the data center may provide the end-user / inventive system with known pathogen / disease mapping information regarding the analysis of biological samples, and subsequently, the inventive system / end-user may transmit assay results to the data center. In addition to assay results, the data center may receive geographic location information and other case-specific information from the end-user / inventive system. The data center may monitor assay results coming from multiple deployed units / inventive systems and utilize pattern detection programs (common in the art). For example, based on data received from multiple end-users, patterns in multiple cases of a given disease in a concentrated geographic area may be detected, or patterns of movement (spread) of a given disease may be detected. Other pattern detection and monitoring related to the onset of specific conditions or pathogens / diseases (e.g., in water supply, food crops, blood banks, etc.) are also considered.

[0274] In this case, the data center may be configured to programmatically generate notifications when threshold patterns are detected. The data center may notify healthcare professionals (ambulances, emergency rooms, doctors), military units, government agencies, etc. Such notifications may be subscription-based, with recipients paying a subscription fee to subscribe to data center alerts and notifications. Different subscription levels may exist, with parties / users paying at one level to have access to the data library and at another level to access the collected assay result data. Another subscription level may provide automated alerts and notifications to subscribers using contact information they provide (where and through what medium / form alerts should be sent).

[0275] Where used herein, notifications and alerts may be in the form of email, telephone, alarm, or other audible and / or visual indications.

[0276] Furthermore, the data center may be configured to provide further responses to the end user after receiving and processing the assay results from the end user. With the given assay results, the data center cross-references other known relevant information, such as genotype and effective treatment / procedure. The data center then transmits this additional information to the end user for field use.

[0277] Therefore, the present invention provides an enhanced method for use in medical care and treatment, disease control, biological defense (pathogen detection at the time of outbreak and in real time, and real-time tracking of infection and contact transmission), and other multi-sector and multi-industry applications across various industries.

[0278] Exemplary device configuration The inventions described herein may comprise or be used in products or devices that include, but are not limited to, handheld devices, computer tablets, notebook computers, smartphones, portable devices, ingestible devices, wearable devices, and injectable devices.

[0279] A “wearable device” is a device intended for use on external parts of the body, such as arms, legs, skin, cheek patches, or torso. Wearable devices are configured to be held on the body, but are not limited to, chip-ons, bracelets, adhesive patches, skin patches, cheek patches, eyeglasses, headbands, ankle bands, shoe accessories (for the soles of the feet), etc. A wearable device may routinely perform the operations of the devices described herein, and the output of the device operations may be interfaced with a computer, such as by wireless information transmission. A wearable device according to an embodiment of the present invention may comprise one or more separate elements, which may be attached together or mechanically interfaced, or not attached together or mechanically interfaced, and / or may reside on one or more different external parts of the body, and may communicate with each other using wireless and / or wired information transmission. A wearable device according to an embodiment of the present invention may also comprise only a portion of such a device as described herein, which may communicate wirelessly and / or wired with other parts of such a device as described herein, which may be inside and / or outside the human body. An exemplary wearable device is shown in Figure 21, comprising (A) a skin patch or dermal patch; (B) a bracelet with a band for holding the device on the neck, arms, legs, or fingers; and (C) a patch on which one side is adhesive and attachable to the outside of the body and the same or opposite side is adhesive and attachable to the device of the present invention. In some embodiments, all elements of the device are contained on the wearable device. In other embodiments, some elements are contained on the wearable device and transmitted to a receiver located elsewhere. A wearable device according to an embodiment of the present invention also comprises only a portion of the device described herein and may communicate wirelessly and / or wired with other parts of such a device according to this specification, the other parts of which are inside and / or outside the human body.

[0280] An “implantable device” is a device that is placed in a surgically or naturally formed cavity in the human body, intended to remain in that location for several days, several months, or longer. The implantable device may routinely perform the operations of the device described herein, and the output of the operations of the device may be interfaced with a computer, such as by wireless information transmission. An implantable device according to embodiments of the present invention may comprise one or more separate elements, which may be attached together or mechanically interfaced, or not attached together or mechanically interfaced, and / or located in one or more different parts of the human body, and which may communicate with each other using wireless and / or wired information transmission. An implantable device according to embodiments of the present invention may also comprise only a portion of such a device according to this specification, which may communicate with other parts of such a device according to this specification wirelessly and / or wired, which may be located inside and / or outside the human body.

[0281] An "ingestible device" is a device that can be delivered orally to a patient for personal monitoring. For example, the device may be an ingestible event marker used with the device described herein to record time and events logged by the patient. The ingestible element is wirelessly connected via information transmission to an external recorder that records the date and time of ingestion as well as the unique serial number of the ingestible device.

[0282] An "injectable device" is a device that can be injected into a part of the human body and can be retained in an intended location within the body. The injectable device may routinely perform the device operations described herein, and the output of the device operations may be interfaced with a computer, such as by wireless information transmission.

[0283] Figure 22 shows an exemplary device relating to an implantable, injectable, or ingestible device. The device of the present invention is incorporated into a carrier substrate.

[0284] Example DNA purification procedure

[0285] The following embodiment will be described with reference to the integrated chip shown in Figure 4 and the fluorescence detection module shown in Figure 9B.

[0286] The user injects the biological sample, magnetic beads, and lysis buffer into the nucleic acid extraction chamber 104 via the sample inlet port 100, and then loads the integration chip 20 into the portable assay system (10, Figure 1). The system incubates the biological sample for 7 minutes, during which lysis occurs and the DNA in the sample binds to the magnetic beads. The system then engages the magnet, thereby clustering the magnetic beads (and bound DNA) in the field area. The beads are washed for 2-3 minutes with the wash buffer injected into the extraction chamber 104 by the user via the sample inlet port 100. The magnet is disengaged during washing. The user repeats this cycle once more before injecting DNA / RNA-free water through the sample inlet port 100. Next, the user disengages the magnet and heats the fluid in the nucleic acid extraction chamber at 70°C for 8-10 minutes, thus releasing the DNA. The magnet engages with the immobilized beads, the reagent addition port 106 is sealed with a passive plug, and the extracted DNA is pushed into the nucleic acid amplification chamber 108 using a syringe.

[0287] The user seals the amplification chamber 108, preferably using a passive plug valve inserted into the reagent addition port 106 and the sample well 120. Once the chamber is sealed, the extracted DNA is mixed with the PCR master mix before the Peltier condenser cycles the temperature between 50°C and 97°C up to 35 times to amplify the extracted DNA. Once amplification is complete, the user unseals the amplification chamber and then pumps the amplified nucleic acid into the detection module using a manual pump syringe connected to the integration chip via the reagent addition port 106.

[0288] The user loads the polymer gel / sieve matrix and CE running buffer into the eight detection channels 112 via the buffer well 116. Next, the molecular weight standard is loaded into the sample well 120. The user then applies 400V to the entire electrode area located on or near the sample well 120 and the sample waste well 118 to draw the amplified nucleic acid into the detection channels 112. Once a sufficient amount of amplified nucleic acid has entered the detection channels, the user switches off the 400V field and then applies a 6kV field from the buffer waste well to the buffer well. This separates the molecular weight standard along the detection channels 112, providing a reference time. The detection channels 112 may then be allowed to flow, after which the user repeats the process using the amplified nucleic acid that was pushed into the sample well 120 and loaded by the pressure of a syringe applied to the nucleic acid amplification chamber 108. This separates the amplified nucleic acid into species and moves it downstream through the detection channels 112.

[0289] As it moves, the molecular weight standard or amplified nucleic acid species passes through the measurement area 1005, where it is investigated under the control of a detection control module 60 configured to detect a fluorescence signal, as shown in Figure 9B. As the species 140 moves through the measurement area 1005, it is irradiated by a laser beam 1004 of an appropriate wavelength, causing the species 140 to emit a fluorescence beam 1010 of a different wavelength. The fluorescence beam 1010 and the laser beam 1004 leave the measurement area 1005 and propagate to a dichroic beam splitter 1007 that reflects the laser beam 1004 and sends the fluorescence beam 1010. The beam block 1008 absorbs the laser beam 1004, and the detector 1012 senses the fluorescence beam 1010. The detector 1012 emits a photocurrent (not shown) balanced with the intensity of the fluorescence beam 1010. The processor 1014 detects, records, and processes the detected photocurrent as a function of time.

[0290] The detected and processed signals are compared against signals from known reference materials analyzed under the same conditions to identify the pathogen in the sample. The system represents this identification process in the output signal graph 1020. By analyzing the known reference materials, a reference signal trace 1021 is obtained with peaks corresponding to known amounts of known pathogens. By comparing the time axis of the signal peak 1022 of the unknown sample with the reference signal trace 1021, the user obtains an indicator of the type of pathogen; the system determines the amount of virus load by calculating the area under the signal peak 1022.

[0291] cell culture

[0292] To demonstrate the performance of the integrated chip, E. coli cells were used for experimental testing. E. coli cells (DH-10B, Invitrogen) were cultured overnight in 5 mL of LB medium (Invitrogen) at approximately 37°C on a 200 rpm stirrer. The stored culture was then either used directly for the experiment or diluted (in water without RNA-degrading enzymes) to obtain the desired cell dilution / concentration. The cultured cell concentration was obtained by measuring the culture using a spectrophotometer (Ultraspec 10, Amersham Biosciences, UK). The details of the E. coli species are as follows: E. coli, DH10B cells (ElectroMAX(trademark) DH10B(trademark), catalog number: 12033-015, Invitrogen, US); genotype: F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZ ΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ - rpsL nupG tonA; the approximate size of the whole genome DNA is about 4.6 × 10⁻¹⁴. 6 It is bp.

[0293] Using these cells, experiments were performed on-chip. Two sets of experiments are described below. In the first set, the stored culture (0.2–2 μL) was used directly for the chip experiment. In the second set of experiments, 1 μL of the stored culture was serially diluted up to infinite dilution. In each of these experiments, measurements of both cell and DNA concentrations were taken during the course of the biological assay, thus enabling quantified data for analysis. Cells were quantified as described above, while DNA enrichment was performed using a Nanovue® (GE Health Sciences) spectrophotometer. However, due to the detection limitations of both spectrophotometers used, quantification of low-concentration samples was difficult until the completion of the amplification-related biological assay, which would allow for easier quantification of DNA amplification. The following experiments were performed on the integrated chip using the protocol described above.

[0294] Identification of pathogens using preserved cell cultures

[0295] Figures 17A–17D and 18 show the results of a first set of experiments in which various amounts of stored cultures were loaded into the extraction chamber of the chip along with magnetic bead solution (i.e., lysis solution and streptavidin-coated Dynal® magnetic bead suspension). The cell count (cfu) entered into the chip was derived from the measured stored concentrate. Following cell lysis and DNA capture with beads as per the protocol above, the DNA was resuspended in 2 μL of water (without RNA-degrading enzymes). The concentration of the extracted DNA was then measured, and the master mix (assay as defined above) was subsequently added.

[0296] Next, amplification was performed in an on-chip amplification chamber.

[0297] Finally, during amplification, the DNA concentration was measured again to induce molecular counting and amplification factors. Figures 17A, 17B, 17C, and 17D show the results of on-chip DNA purification and amplification. The plots show: Figure 17A - amplification factor as a function of start DNA amount, Figure 17B - final DNA amount as a function of start DNA amount, Figure 17C - final DNA concentration as a function of start DNA concentration, and Figure 17D - amount of extracted DNA as a function of input cell counting.

[0298] Conventional gel-based electrophoresis, as shown in Figure 18, allowed for confirmation of the resulting amplification product size at approximately 700 bp, which was predicted from the initial primer design. The results shown in Figure 18 are as follows:

[0299] Lane 2: DNA Ladder;

[0300] Lane 4: Positive control group for DH10B cell amplification (approximately 700 bp);

[0301] Lane 5: Negative control group for DH10B cell amplification;

[0302] Lane 6: DNA Ladder;

[0303] Lane 8: Positive control group for DH10B cell amplification (approximately 700 bp);

[0304] Lane 9: Negative control group for DH10B cell amplification; Lane 10: 50 bp ladder.

[0305] Identification of pathogens using cell culture dilution Figures 19A–19D and 20 show the results of a second experimental set in which 1 μL of the preserved culture solution was serially diluted with 9 μL of water to achieve cell dilutions containing fewer than one cell at most. Because the tip-based approach can only accommodate very small volumes (typically a few microliters), the culture dilutions were then centrifuged to ensure the use of all cells in the dilution. These centrifuged cell pellets were then added to 10 μL of Dynal® magnetic bead solution and subjected to an extraction process similar to that of the first experimental set.

[0306] As previously performed, the extracted DNA was resuspended in 2 μL of water, and then approximately 8 μL of amplification master mix was added. Amplification was then carried out on-chip.

[0307] DNA concentration was measured before and after amplification. The data was then used to derive DNA concentration and amplification factors for each culture dilution sample used. Figures 19A, 19B, 19C, and 19D show the results of on-chip DNA purification and amplification. The plots show: Figure 19A - the number of DNA molecules measured as a function of the number of input cells; Figure 19B - the final number of DNA molecules before and after amplification as a function of the number of DNA molecule starts; Figure 19C - the amplification factor as a function of the number of DNA molecule starts; and Figure 19D - the amplification factor as a function of the theoretical number of DNA molecule starts.

[0308] Using conventional laboratory-based gel electrophoresis, the resulting amplification product size was confirmed to be approximately 700 bp, as shown in Figure 20. The results shown in Figure 20 are as follows:

[0309] Lane 1: DNA Ladder;

[0310] Lane 3: Positive control group for DH10B cell amplification (approximately 700 bp);

[0311] Lane 5: Negative control group for DH10B cell amplification;

[0312] Lane 7: DNA Ladder;

[0313] Lane 9: Positive control group for DH10B cell amplification (approximately 700 bp).

[0314] result

[0315] As is evident from any of the amplification experiments above, efficiency decreases as the concentration of the starting sample increases (i.e., as suggested by the amplification factor). In an ideal assay where theoretical limiting factors are negligible, exponential amplification should be expected. However, given limitations such as (1) loss of enzyme efficiency after repeated cycles, (2) lack of sufficient dNTPs and primer sets, and (3) adsorption factors that inhibit the recovery of amplified DNA or cause insufficient inhibition of amplified DNA (which are conversely enhanced by the on-chip surface-to-volume ratio), achieving the theoretical amplification amount may be difficult. However, as noted in the second experimental set, detectable amplification is achieved with only about 12 starting cells in the uncontaminated input sample.

[0316] Demonstration of HIV-1 amplification and quantification

[0317] Unlike commercially available PCR machines for viral load studies, the system according to the embodiments of the present invention requires no offline sample preparation whatsoever, with the processing steps integrated within a customized nanochip. The biofluid is applied directly to the nanochip contained within the device.

[0318] The systems according to embodiments of the present invention relate to the quantity of cDNA, DNA, or RNA over a very wide operating range. For example, in an assay for inactivated HIV-1 cDNA, the higher the HIV-1 titer of the sample, the sooner the detection signal exceeds the baseline value. The lower the HIV-1 titer of the sample, the more amplification cycles are required to produce a detectable amount of HIV-1 material. The appearance of the detector signal is reported as the critical threshold (Ct). Ct is defined as the fraction of cycles at which the detector signal exceeds a given threshold and begins an exponential growth phase. A higher Ct value indicates a lower threshold for initiating HIV-1 target material.

[0319] Figure 23 shows target standard curves for a dilution series ranging from 10⁶ HIV-1 to 10⁻² HIV-1 cDNA molecules in experiments according to embodiments of the present invention. As the concentration of HIV-1 material increases, the standard curves show a shift to faster cycles. Thus, the leftmost standard curve corresponds to the highest viral titer level, while the rightmost standard curve corresponds to the lowest viral titer level (Figure 23). The horizontal blue line represents the Ct value for this experimental set. Pilot experiments in embodiments of the present invention demonstrate an operating range for HIV detection from less than 50 copies to 2 million copies of starting HIV-1 cDNA or RNA molecules, and demonstrate proof of concept with other pathogens. Experiments are underway to replicate the single molecular weight detection already achieved with another pathogen (E. coli). In the upper left corner of Figure 23 is a drawing of a prototype device according to embodiments of the present invention, which is smaller than a conventional laptop computer. Figure 26 shows a larger view of the device, illustrating the display screen 2601 and the compartment 2602 into which a chip containing a biological sample for analysis is inserted. The device's operating system is intuitive to the user and provides seamless functionality, along with innovative integration capabilities for data sharing and storage. The data in Figure 23 was prepared within one hour.

[0320] A device according to an embodiment of the present invention has been shown to produce a standard curve for quantification starting from an armored HIV-1 RNA virus reference sample containing precisely quantified copy number HIV-1 RNA virus sequence targets. Armored RNA control has become a standard industry practice in a wide range of diagnostic tests, encompassing many commercially available HIV virus load tests.

[0321] Direct comparison of FDA-approved Stratagene PCR machines

[0322] The prototype was benchmarked against the most standard quantitative PCR machine, the Stratagene MX4000, to further demonstrate the system feasibility data according to the embodiments of the present invention.

[0323] In Figure 24, inactivated HIV-1 was serially diluted to starting amounts ranging from 10⁶ to 10² HIV-1 cDNA molecules. Viral load was quantified using threshold cycles (Ct) from standard amplification curves prepared by SYBR fluorescence. These standard curves were reproduced based on known HIV-1 viruses. Figure 24 shows the operating range of a conventional PCR machine for amplifying target HIV-1. The horizontal blue line reflects the Ct values ​​for this experimental set. Figure 23 (top) shows the same sample with known concentrations of HIV-1 in a system according to an embodiment of the present invention. These data provide conceptual evidence that the quantitative viral load data from the embodiment according to the present invention is conceptually equivalent to that of a Stratagene system based on Ct, curve fitting, and operating range. Compared to the system according to the embodiment of the present invention, the most standard FDA-approved viral load Stratagene machine is approximately 10 times larger.

[0324] Detection of bacterial loads with precise quantification

[0325] The common bacterial pathogen E. coli was tested using a system according to the embodiments of the present invention, demonstrating the operational range for detecting 10,000,000 E. coli DNA / ul from 1,000 copies (Figure 25A, left). The Pearson correlation coefficient R² = 0.9886 shows a statistically significant linear fit between the quantification cycle and DNA copy number, demonstrating the fidelity of the technique (Figure 25B, right).

[0326] All patents, published applications, and teachings referenced herein are incorporated herein by reference.

[0327] Although the present invention has been specifically illustrated and described with reference to exemplary embodiments, it will be understood by those skilled in the art that various modifications can be made in form and detail without departing from the scope of the invention as encompassed in the accompanying claims.

Claims

1. A system for analysis of a biological sample, the system comprising: a mobile device housing an integrated chip for analyzing a loaded biological sample, the mobile device and the integrated chip both comprising an extraction module, an optional amplification module, and a detection module; Control mechanism and Equipped with During operation of the system, the mobile device and the integrated chip are both configured to perform at least one cycle of operations, the operations including: (a) extracting an analyte from the biological sample using the extraction module; (b) optionally generating one or more products from the analyte using the amplification module; and (c) detecting the analyte or one or more products generated from the analyte using the detection module; The integrated chip and the mobile device employ a control mechanism to selectively apply nanoscale control during at least one cycle of the operations (a)-(c).

2. The system described in claim 1, wherein the integrated chip and the mobile device use the control mechanism to selectively apply the nanoscale control during step (a), which is at least one cycle of the operations (a) to (c).

3. The system described in claim 1, wherein the integrated chip and the mobile device use the control mechanism to selectively apply the nanoscale control during step (c), which is at least one cycle of the operations (a) to (c).

4. The system of claim 1, wherein applying the nanoscale control includes at least one of applying hydrodynamic stress to at least one of the analyte and one or more of the products, and hydrodynamically focusing the analyte and at least one of the products.

5. The system of claim 1, wherein applying the nanoscale control includes adjusting the rotational orientation of at least one of the analyte and one or more of the products.

6. The system of claim 1, wherein applying the nanoscale control includes applying at least one of electromagnetic tension and mechanical force to at least one of the analyte and one or more of the products.

7. The system of claim 1, wherein applying the nanoscale control includes adjusting at least one environmental parameter associated with at least one of the analyte and one or more of the products on a length scale of 100 nm or less.

8. The system of claim 7, wherein at least the environmental parameters are selected from the group consisting of a temperature of at least one of the specimen and one or more of the products, a pressure applied to the specimen and at least one of the products, and a concentration of at least one reagent combined with the specimen and at least one of the products.

9. The system described in claim 1, wherein the mobile device is equipped with a data processor configured to control the extraction, amplification, and detection operations of the operation cycle.

10. The system of claim 1, wherein detecting the sample, or one or more of the products produced from the sample, includes determining at least a portion of a nucleic acid sequence of the sample, or one or more of the products produced from the sample.

11. A system for analyzing a sample, comprising:

1. A fluidic chip configured to perform a cycle of operations, the cycle of operations comprising: (a) extracting biological material from the sample in an extraction control module; (b) producing one or more products from the biomaterial in an amplification control module; (c) detecting one or more products in a detection control module; A fluidic chip comprising: a device configured to interface with the fluidic chip, the device including a processor configured to operate one or more of the control modules during cycles (a)-(c) of operation; Equipped with the control module is configured to provide nanoscale control during the operation cycles (a)-(c); The nanoscale control includes: applying a stress to a molecular species; adjusting the rotational orientation of molecular species; Adjusting the translational position of molecular species A system comprising at least one of:

12. The system described in claim 11, wherein the stress is hydrodynamic stress.

13. The system of claim 11, wherein applying the stress to the molecular species comprises hydrodynamically focusing the molecular species.

14. The system of claim 11, wherein adjusting the rotational orientation of the molecular species comprises applying a hydrodynamic force to the molecular species.

15. The system of claim 11, wherein applying the stress to the molecular species comprises applying at least one of an electromagnetic tension and a mechanical force to the molecular species.

16. The system of claim 11, wherein the nanoscale control further comprises adjusting at least one environmental parameter associated with the molecular species on a length scale of 100 nm or less.

17. The system of claim 16, wherein at least one of the environmental parameters is selected from the group consisting of a temperature of the molecular species, a pressure applied to the molecular species, and a concentration of at least one reagent combined with the molecular species.

18. The system of claim 11, wherein detecting one or more of the products includes determining at least a portion of a nucleic acid sequence of one or more of the products.

19. A system for determining the nucleic acid load of a sample, the system comprising: a fluidic chip including a plurality of channels; a device configured to house the fluidic chip, the device comprising a processor and a detector coupled to the processor; one or more fluid flow regulators disposed on the fluidic chip or within the device such that when the fluidic chip is housed by the device, the processor is coupled to the fluidic flow regulators; Equipped with the detector is configured to generate a signal representative of nucleic acids of the sample; The processor includes instructions that, when executed by the processor, cause the processor to: controlling fluid flow within the fluidic chip to adjust at least one of rotational, vibrational, and translational degrees of freedom of the nucleic acids of the sample within the multiple channels; The system quantitatively determines the nucleic acid load of the sample based on the measured signal.

20. The system described in claim 19, wherein the nucleic acid is derived from a pathogen and the nucleic acid load is the pathogenic load of the sample.

21. The system of claim 20, wherein the pathogenic load is a viral or bacterial load of the sample.

22. The system of claim 19, wherein the nucleic acid corresponds to a biomarker of the sample and the nucleic acid load is a biomarker concentration in the sample.

23. The system of claim 19, wherein adjusting at least one of the rotational, vibrational, and translational degrees of freedom of the nucleic acid comprises applying a hydrodynamic stress to the nucleic acid.

24. The system of claim 19, wherein adjusting at least one of the rotational, vibrational, and translational degrees of freedom of the nucleic acid comprises hydrodynamically focusing the nucleic acid.

25. The system described in claim 19, wherein the processor further includes instructions which, when executed by the processor, cause the processor to adjust at least one environmental parameter associated with the nucleic acid on a length scale of 100 nm or less.

26. The system described in claim 25, wherein at least one of the environmental parameters is selected from the group consisting of a temperature of the nucleic acid, a pressure applied to the nucleic acid, and a concentration of at least one reagent combined with the nucleic acid.

27. The system of claim 19, wherein determining the nucleic acid loading of the sample based on the measured signal includes determining an area under the measured signal.

28. A method for determining the nucleic acid load of a sample, the method comprising: (a) extracting nucleic acid from a sample and depositing said nucleic acid in one channel of a plurality of channels on a fluidic chip; (b) amplifying said nucleic acid to generate a population of amplicons of said nucleic acid; (c) measuring a detection signal representative of the population of amplicons; During at least one of steps (a)-(c), controlling fluid flow within one of said plurality of channels to modulate at least one of rotational, vibrational, and translational degrees of freedom of at least one of said nucleic acids and said amplicons; and quantitatively determining the nucleic acid loading of the sample based on the measured detection signal.

29. The method of claim 28, wherein the nucleic acid is derived from a pathogen and the nucleic acid load is the pathogenic load of the sample.

30. The method of claim 29, wherein the pathogenic load is a viral or bacterial load of the sample.

31. The method of claim 28, wherein the nucleic acid corresponds to a biomarker in the sample and the nucleic acid load is a biomarker concentration in the sample.

32. The method of claim 28, wherein the step of adjusting at least one of the rotational, vibrational, and translational degrees of freedom of the nucleic acid comprises applying a hydrodynamic stress to the nucleic acid.

33. The method of claim 28, wherein the step of adjusting at least one of the rotational, vibrational, and translational degrees of freedom of the nucleic acid comprises hydrodynamically focusing the nucleic acid.

34. The method of claim 28, further comprising adjusting at least one environmental parameter associated with the nucleic acid on a length scale of 100 nm or less.

35. The method of claim 34, wherein at least one of the environmental parameters is selected from the group consisting of a temperature of the nucleic acid, a pressure applied to the nucleic acid, and a concentration of at least one reagent combined with the nucleic acid.

36. The method of claim 28, wherein the step of determining the nucleic acid loading of the sample based on the measured signal includes determining an area under the measured signal.

37. A system for detecting a protein in a sample, the system comprising: a fluidic chip including a plurality of channels; a device configured to house the fluidic chip, the device comprising a processor and a detector coupled to the processor; one or more fluid flow regulators disposed on the fluidic chip or within the device such that when the fluidic chip is housed by the device, the processor is coupled to the fluidic flow regulators; Equipped with The processor includes instructions that, when executed by the processor, cause the processor to: During at least one of extracting proteins from a sample and detecting proteins, controlling a flow of fluid within the fluidic chip to adjust at least one of rotational, vibrational, and translational degrees of freedom of proteins of the sample within the multiple channels; The system detects the protein based on a measurement signal generated by the detector.

38. The system of claim 37, wherein adjusting at least one of the rotational, vibrational, and translational degrees of freedom of the protein comprises applying a hydrodynamic stress to the protein.

39. The system of claim 37, wherein modulating at least one of the rotational, vibrational, and translational degrees of freedom of the protein comprises hydrodynamically focusing the protein.

40. The system of claim 37, wherein the processor further includes instructions which, when executed by the processor, cause the processor to adjust at least one environmental parameter associated with the protein on a length scale of 100 nm or less.

41. The system of claim 40, wherein at least one of the environmental parameters is selected from the group consisting of a temperature of the protein, a pressure applied to the protein, and a concentration of at least one reagent combined with the protein.

42. A method for detecting a protein in a sample, the method comprising: (a) extracting proteins from a sample and depositing the proteins in one of a plurality of channels on a fluidic chip; (b) measuring a detection signal representative of said protein; During at least one of steps (a)-(b), controlling fluid flow within one of the plurality of channels to modulate at least one of rotational, vibrational, and translational degrees of freedom of the protein; detecting the protein based on the measured detection signal; The method according to claim 1, 43. The method of claim 42, wherein the step of adjusting at least one of the rotational, vibrational, and translational degrees of freedom of the protein comprises applying a hydrodynamic stress to the protein.

44. The method of claim 42, wherein the step of adjusting at least one of the rotational, vibrational, and translational degrees of freedom of the protein comprises hydrodynamically focusing the protein.

45. The method of claim 42, further comprising adjusting at least one environmental parameter associated with the protein on a length scale of 100 nm or less.

46. ​​The method of claim 45, wherein at least one of the environmental parameters is selected from the group consisting of a temperature of the protein, a pressure applied to the protein, and a concentration of at least one reagent combined with the protein.

47. A system comprising: a fluidic chip including a plurality of channels; a device configured to house the fluidic chip, the device comprising a processor and a detector coupled to the processor; one or more fluid flow regulators disposed on the fluidic chip or within the device such that when the fluidic chip is housed by the device, the processor is coupled to the fluid flow regulators; The processor includes instructions that, when executed by the processor, cause the processor to: Controlling fluid flow within the fluidic chip to modulate at least one of rotational, vibrational, and translational degrees of freedom of a plurality of analytes of a sample within the plurality of channels during at least one set of operations, the at least one set of operations comprising: (a) extracting a plurality of said analytes from said sample; (b) amplifying one or more of the plurality of analytes to generate a population of one or more amplicons for one or more of the plurality of analytes; (c) detecting a plurality of said populations of analytes and amplicons; Including, receiving from the detector generated measurement signals representative of the plurality of analytes; identifying a plurality of biomarkers in the sample based on the measurement signals; The system integrates a plurality of said biomarkers into a personalized medical profile associated with said sample.

48. A method comprising: (a) extracting a plurality of analytes from a sample and depositing the plurality of analytes in one of a plurality of channels on a fluidic chip; (b) optionally amplifying one or more of the plurality of said analytes to generate one or more populations of amplicons for one or more of the plurality of said analytes; (c) measuring detection signals representative of a plurality of said analytes and said population of amplicons; During at least one of the operations (a)-(c), controlling a flow of fluid within one of the plurality of channels to adjust at least one of rotational, vibrational, and translational degrees of freedom of the plurality of analytes; identifying a plurality of biomarkers in the sample based on the measured detection signals; combining the plurality of biomarkers into a personalized medical profile associated with the sample; The method according to claim 1,

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