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

Abstract

To provide an automated, integrated, compact, robust, rapid, accurate and easy-to-use device and method for detecting, quantifying and identifying nucleic acids.SOLUTION: Provided is a portable or 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 combination with a portable system or a mobile device for analyzing detected signals, and for comparing and distributing the results via a wireless network.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 March 11, 2014; and claims the benefit of U.S. Provisional Patent Application No. 61 / 875,661, filed September 9, 2013; and claims the benefit of U.S. Provisional Patent Application No. 61 / 790,354, filed March 15, 2013. The entire teachings of the above provisional patent applications are incorporated herein by reference. [Background technology]

[0002] 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 cosmeceuticals, personalized nutraceuticals, companion diagnostics, drug monitoring, pharmacogenetics and nutrigenomics. The identity of a species in a biological sample can be confirmed by comparing the nucleic acids present in the sample with those of a known reference sample. However, before this comparison can be made, the nucleic acids must be extracted from the sample, amplified and then detected. Typically, the extraction, amplification and detection steps are carried out over a period of hours, days or weeks in a laboratory or hospital. For example, amplification is typically associated with polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,202 and 4,683,195. To amplify nucleic acids using conventional PCR, the nucleic acids 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 performed in a mobile or field environment outside of specialized laboratory facilities. Extraction and amplification alone can take hours, if not days, depending on the type of organism, the length of the nucleic acid strand and the number of cycles. Furthermore, commercially available devices and methods require skilled labor, running water and electricity. Furthermore, temperature, pH and buffer composition must be tightly controlled. Contaminants can inhibit or interfere with the nucleic acid polymerase enzymes used in the replication, reducing the efficiency and fidelity of the amplification process. Similar limitations apply to conventional techniques for extracting and detecting nucleic acids. Thus, there is a need for automated, integrated, compact, robust, rapid, accurate and easy-to-use devices and methods for detecting, quantifying and identifying nucleic acids. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Pat. No. 4,683,202 [Patent Document 2] U.S. Pat. No. 4,683,195 Summary of the Invention

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

[0006] The present invention provides a portable or 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 together with a portable system or mobile device for detection signal analysis, and comparing and providing the results via a wireless network. Interchangeable modules can be used for one or more of extraction, amplification, and detection.

[0007] The present invention has several advantages. The present invention 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 take hours or weeks to characterize or analyze even large samples, the present invention typically produces measurements within an hour, or even 30 minutes, for example, within minutes. The present invention is sensitive enough 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 a portable or mobile device that uses a compact integrated chip that may be disposable. Results can be provided 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 knowledgeable technicians and can function outside of a hospital or centralized laboratory. 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 may be used to detect and differentiate 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 sensitive enough to differentiate species within a genus, or to differentiate point mutations and / or biomarker sequences and / or disease susceptibility alleles. In embodiments of the present invention using disposable integrated chips, there are also significant cost savings compared to the costs of existing diagnostic assays.

[0009] Additionally, in embodiments of the invention that utilize the non-thermal nucleic acid amplification process disclosed in U.S. Pat. No. 7,494,791, the disclosure of which is incorporated herein by reference in its entirety, approximately 1×10 -7 Less than 10 errors or better (e.g., 10 per base pair) -10Error rates of less than 1×10 6 errors per base pair can be achieved, even for "difficult sequences" (sequences where the polymerase enzyme tends to slip, make errors, or stop working; examples of difficult sequences include repetitive sequences, poly-A sequences, GC-rich sequences, trinucleotide repeat sequences, etc.). -3 Less than 10 errors or better (e.g., 10 per base pair) -6 Error rates of less than 100 errors can be achieved. Utilizing reagents capable of surviving more than 100 cycles of nucleic acid replication, sequences up to 20,000 base pairs in length can be amplified using the disclosed non-thermal nucleic acid amplification methods. Other isothermal methods of amplification can be used, such as the Loop Mediated Isothermal Amplification (LAMP) technique, such as that described in T. Notomi, et al., Nucleic Acids Research, 28, e63 (2000); the Helicase-Dependent Amplification (HDA) technique, such as that described in Vincent M, Xu Y, Kong H. (2004), "Helicase-dependent isothermal DNA amplification," EMBO Rep 5 (8): 795-800; the Strand Displacement Amplification (SDA) technique, such as that described in GT Walker, et. al., Proc. Natl. Acad. Sci USA, 89, 392-396 (1992); the teachings of all of which are incorporated herein by reference in their entirety. Bridge, rolling circle and any other amplification method (thermal or isothermal) can be used.

[0010] In an embodiment according to the present invention, a system for rapid analysis of biological samples is provided. The system comprises a mobile device that receives at least one integrated chip. The mobile device processes the integrated chip and analyzes the biological sample loaded thereon. The mobile device and the integrated chip together are configured to perform at least one of the manipulation and control of the molecules or fluidic systems on the integrated chip. The mobile device and the integrated chip together are configured to precisely control at least one parameter governing at least one of a plurality of steps of the analysis of the 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; may comprise a nucleic acid amplification module in fluid communication with the extraction module; and comprises a biological sample detection module in fluid communication with the nucleic acid amplification module or the extraction module. The portable control assembly processes the integrated chip and analyzes the biological sample loaded thereon by using: the extraction control module; the nucleic acid amplification control module operatively linked to the extraction control module; and the biological sample detection control module operatively linked to the nucleic acid amplification module and the extraction module.

[0012] 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 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 in fluid communication with the extraction module; and a detection module in fluid communication with the nucleic acid amplification module. The portable assembly comprises an extraction control module, which in some embodiments comprises a magnetic particle capture means for capturing magnetic particles introduced into the biological sample loaded onto the integrated chip; a nucleic acid amplification control module operatively linked to the extraction control module, which in some embodiments comprises a heating means for heating / cooling the nucleic acid amplification module of the integrated chip, e.g., thermoelectric, thin film, infrared or optical based or mechanical; and a detection control module operatively linked to the nucleic acid amplification module and the nucleic acid extraction module. In some embodiments, the detection control module comprises: a fluorescence detection means for detecting nucleic acids and proteins; and in some embodiments, a capillary electrophoresis (CE) control means, e.g., operably linked to the fluorescence detection means, said CE control means further comprising a high voltage control for applying a voltage across the nucleic acid detection module of the integrated chip that is sufficient to effect separation of nucleic acids or proteins; and a fluid pressure generating means for moving the biological sample and / or nucleic acids through the integrated chip.

[0013] In another embodiment according to the present invention, a method for rapid analysis of biological samples is provided.The method includes: (1) preparing at least one integrated chip, which comprises a nucleic acid extraction module; a nucleic acid amplification module that is fluidly connected to the nucleic acid extraction module; and a nucleic acid detection module that is fluidly connected to the nucleic acid amplification module; (2) loading at least one biological sample onto the at least one integrated chip; (3) functionally connecting a portable control assembly to the at least one integrated chip, which comprises a nucleic acid extraction control module; a nucleic acid amplification control module that is functionally connected to the nucleic acid extraction control module; and a nucleic acid detection control module that is functionally connected to the nucleic acid amplification module and the nucleic acid extraction module; and (4) operating the portable control assembly to carry out extraction, amplification and detection of nucleic acid from the biological sample loaded onto the integrated chip.

[0014] In another embodiment according to the present invention, a method for rapid analysis of biological samples is provided, the method includes: (1) providing at least one integrated chip including a protein extraction module and a protein detection module in fluid communication with the protein extraction module; (2) loading at least one biological sample onto the at least one integrated chip; (3) operatively connecting a portable control assembly to the at least one integrated chip, the portable control assembly including a protein extraction control module and a protein detection control module operatively connected with the protein extraction module; and (4) operating the portable control assembly to perform 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 acid from biological samples.The integrated chip comprises a housing in which microfluidic channels are integrated, which are fluidically connected to a nucleic acid extraction module, a nucleic acid amplification module and a nucleic acid separation module in series; at least one sample inlet port is fluidically connected to the nucleic acid extraction module for injecting biological samples and reagents; where the nucleic acid extraction module comprises at least one extraction chamber for extracting nucleic acid from biological samples, and the extraction chamber is connected to the sample inlet port by at least one sample transport channel; where the nucleic acid amplification module comprises at least one amplification chamber for amplifying nucleic acid, and the nucleic acid amplification chamber is connected to the extraction chamber by at least one nucleic acid transport channel; and where the nucleic acid separation module comprises at least one detection channel for separating and detecting nucleic acid, and the detection channel is connected to the nucleic acid amplification chamber by at least one amplification product transport channel.

[0016] In another embodiment according to the present invention, a method for rapid analysis of biological samples is provided. The method includes preparing at least one integrated chip, the integrated chip includes: 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. The method further includes loading at least one biological sample onto the at least one integrated chip; and operably linking a portable control assembly to the at least one integrated chip. The portable control assembly includes a nucleic acid extraction control module; a nucleic acid amplification control module in operably linked with the nucleic acid extraction control module; and a nucleic acid detection control module in operably linked with the nucleic acid amplification module and the nucleic acid extraction module. The method further includes operating the portable control assembly to perform extraction, amplification and detection of nucleic acid from the biological sample loaded onto the integrated chip; and determining at least one biomarker associated with the person from whom the at least one biological sample is sourced based on detection of nucleic acid from the biological sample.

[0017] In further related embodiments, the method may include determining, based on the at least one biomarker, at least one of: (i) a dosage of at least one agent to effect therapeutic treatment of a disease condition associated with the at least one biomarker; (ii) a combination of agents to effect therapeutic treatment of a disease condition associated with the at least one biomarker; and (iii) a determination of whether a person from whom the at least one biological sample is derived will respond to agent treatment for a disease condition associated with the at least one biomarker.

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

[0019] In further related embodiments, the method may include determining at least one biomarker associated with the person who is the source of the at least one biological sample based on detection of nucleic acid from the biological sample; and determining, based on the at least one biomarker, at least one of: (i) selecting a personal care product for the person who is the source of the at least one biological sample; (ii) a delivery amount of the personal care product for the person; (iii) e.g., a cosmetic skin type for the person; and (iv) an amount of the at least one cosmetic biomarker for the person in the at least one biological sample.

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

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

[0022] In other related embodiments, the method may include determining at least one biomarker associated with the person from whom the at least one biological sample is derived based on detection of nucleic acid from the biological sample; and determining whether the person from whom the at least one biological sample is derived is a member of the subset genotype with respect to the at least one biomarker based on the at least one biomarker.

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

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

[0025] In another embodiment according to the present invention, a system for rapid analysis of biological samples is provided. The system comprises at least one integrated chip, the integrated chip comprises: 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 in functional communication with the nucleic acid extraction control module; and a nucleic acid detection control module in functional communication with the nucleic acid amplification module and the nucleic acid extraction module, the portable control assembly performs extraction, amplification and detection of nucleic acid from the biological sample loaded onto the integrated chip. The system further comprises a genetic analyzer configured to determine at least one biomarker associated with the person who is the source of the at least one biological sample based on the detection of nucleic acid from the biological sample. The genetic analyzer is further configured to determine at least a portion of an individualized genomic profile of the 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 biological samples is provided. The system comprises at least one integrated chip, the integrated chip comprises: 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 in functional communication with the nucleic acid extraction control module; and a nucleic acid detection control module in functional communication with the nucleic acid amplification module and the nucleic acid extraction module, the portable control assembly performs extraction, amplification and detection of nucleic acid from the biological sample loaded onto the integrated chip. The system further comprises a genetic analysis unit configured to determine at least one biomarker associated with the person from whom the at least one biological sample is sourced based on the detection of nucleic acid from the biological sample; and a cardiac control unit connected to the genetic analysis unit and configured to control an enhanced extracorporeal counterpulsation (EECP) or other cardiac control unit 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] The foregoing will become apparent from the following more particular description of illustrative embodiments of the invention, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the various views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the invention. [Brief description of the drawings]

[0030] [Figure 1] FIG. 1 illustrates one embodiment of a portable assay or mobile system with a compact integrated chip for detecting and analyzing nucleic acids. [Figure 1A]FIG. 1A illustrates an embodiment of the modular design of a portable assay system in communication with a genomic database. [Diagram 2] FIG. 2 is an illustration of an embodiment of a modular design of the systems and devices of the present invention. [Figure 2A] FIG. 2A shows an embodiment of a modular design of the extraction control module. [Figure 2B] FIG. 2B shows an embodiment of a modular design of the amplification control module. [Diagram 3] FIG. 3 is a diagrammatic example of a flow chart of a method for detecting and analyzing nucleic acids. [Figure 4] FIG. 4 is an illustration of an embodiment of a compact integrated chip that may be used with the devices and methods of the present invention. [Diagram 5] FIG. 5 illustrates an exemplary passive plug for controlling fluid flow on an integrated chip. [Figure 6A] FIG. 6A illustrates an exemplary electromagnetically controllable valve and method for controlling fluid flow through an integrated chip utilized in the present invention. [Figure 6B] FIG. 6B illustrates an exemplary electromagnetically controllable valve and method for controlling fluid flow through an integrated chip utilized in the present invention. [Figure 7A] FIG. 7A illustrates an exemplary method for controlling fluid flow to and from on-chip wells. [Figure 7B] FIG. 7B illustrates an exemplary method for controlling fluid flow to and from on-chip wells. [Figure 8A] FIG. 8A shows an exemplary arrangement of channels and a method for injecting a sample into the channels used for capillary electrophoresis according to an embodiment of the invention. [Figure 8B] FIG. 8B shows an exemplary arrangement of channels and a method for injecting a sample into the channels used for capillary electrophoresis according to an embodiment of the present invention. [Figure 8C]FIG. 8C shows an exemplary arrangement of channels and a method for injecting a sample into the channels used for capillary electrophoresis according to an embodiment of the invention. [Figure 9A] FIG. 9A shows an embodiment of a modular design of the fluorescence detection control module. [Figure 9B] FIG. 9B illustrates an exemplary system and method for detecting fluorescent signals generated by a nucleic acid detection module of an integrated chip. [Figure 10] FIG. 10 is a flow chart illustrating the utility of the present invention in various applications. [Figure 11] FIG. 11 illustrates a hardware module for use with a compact integrated chip according to an embodiment of the present invention. [Figure 12] FIG. 12 illustrates a Peltier heating device for use with the hardware module of FIG. [Figure 13] FIG. 13 shows a detection system for use with the compact integrated chip. [Figure 14] FIG. 14 illustrates a hardware system for use with a compact integrated chip according to an embodiment of the present invention. [Figure 15A] FIG. 15A shows a different view of an expandable encapsulated elastic membrane-based compact integrated chip microfluidic valve. [Figure 15B] FIG. 15B shows a different view of the expandable encapsulated elastic membrane-based compact integrated chip microfluidic valve. [Figure 16A] FIG. 16A shows a different view of an expandable encapsulated elastic membrane-based compact integrated chip microfluidic valve assembly. [Figure 16B] FIG. 16B shows a different view of the expandable encapsulated elastic membrane-based compact integrated chip microfluidic valve assembly. [Figure 16C] FIG. 16C shows a different view of the expandable encapsulated elastic membrane-based compact integrated chip microfluidic valve assembly. [Figure 16D]FIG. 16D shows a different view of the expandable encapsulated elastic membrane-based compact integrated chip microfluidic valve assembly. [Figure 16E] FIG. 16E shows a different view of the expandable encapsulated elastic membrane-based compact integrated chip microfluidic valve assembly. [Figure 16F] FIG. 16F shows a different view of the expandable encapsulated elastic membrane-based compact integrated chip microfluidic valve assembly. [Figure 17A] FIG. 17A shows the results of on-chip DNA purification and amplification. [Figure 17B] FIG. 17B shows the results of on-chip DNA purification and amplification. [Figure 17C] FIG. 17C shows the results of on-chip DNA purification and amplification. [Figure 17D] FIG. 17D shows the results of on-chip DNA purification and amplification. [Figure 18] FIG. 18 is a depiction of an agarose gel-based electrophoresis confirming the size of the resulting amplification products. [Figure 19A] FIG. 19A shows the results of on-chip DNA purification and amplification. [Figure 19B] FIG. 19B shows the results of on-chip DNA purification and amplification. [Figure 19C] FIG. 19C shows the results of on-chip DNA purification and amplification. [Figure 19D] FIG. 19D shows the results of on-chip DNA purification and amplification. [Figure 20] FIG. 20 is a depiction of an agarose gel-based electrophoresis confirming the size of the resulting amplification products. [Figure 21] Figure 21 shows wearable devices: (A) a skin or dermal patch; (B) a bracelet comprising a device of the invention and a band for holding the device on the wrist, arm, leg, finger; and (C) a patch with one side adhesively attachable to the exterior of the body and the same or opposite side adhesively attachable to a device of the invention. [Figure 22]FIG. 22 illustrates a device configured as an implantable, injectable or ingestible device of the present invention. [Diagram 23] FIG. 23 is a graph of a target standard curve for a dilution series for HIV quantification in an experiment according to an embodiment of the present invention. [Figure 24] FIG. 24 is a graph of curves illustrating the typical operating range of a conventional PCR machine for amplifying the target HIV-1. [Figure 25A] FIG. 25A is a graph of curves showing testing of the common bacterial pathogen E. Coli in a system according to an embodiment of the present invention. [Figure 25B] FIG. 25B is a graph of a linear fit between quantitative cycles and DNA copy number for the experiment in FIG. 25A. [Figure 26] FIG. 26 is a device diagram according to an embodiment of the present invention. [Figure 27] FIG. 27 is a table of selected cardiovascular inflammatory biomarkers that may be determined, measured and / or monitored according to embodiments of the present invention. [Figure 28] FIG. 28 is a table of selected diabetic inflammatory biomarkers that may be determined, measured and / or monitored according to embodiments of the present invention. [Figure 29] FIG. 29 is a table of selected biomarkers for the progression of obesity, diabetes and cardiovascular disease that may be determined, measured and / or monitored according to embodiments of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 devices and / or methods that operate with or relate to fluid volumes between 0.1-100 μL and preferably between 1 and 10 μL.

[0034] As used herein, "fluidic system" refers to a system that allows fluid to flow, e.g., a system that allows fluid to flow in at least one channel, in at least one chamber, in at least one well and / or in at least one port, each of which may be macrofluidic.

[0035] "Nucleic acid" as used herein refers to a macromolecule composed of a chain (polymer or oligomer) of monomeric nucleotides. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It is further understood that the present invention may be used to detect and identify samples containing artificial nucleic acids such as peptide nucleic acid (PNA), morpholino, locked nucleic acid (LNA), glycol nucleic acid (GNA) and threose nucleic acid (TNA), among others. In various embodiments of the present invention, the nucleic acid may be from a variety of sources, such as bacteria, viruses, human and animals, and sources such as plants and fungi, among others. The source may be a pathogen. Or the source may be a synthetic organism. The nucleic acid may be genomic, extrachromosomal or synthetic. Where the term "DNA" is used herein, one of skill in the art will understand that the methods and devices described herein may be applied to other nucleic acids, such as RNA or the above. Additionally, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used herein to encompass polymeric forms of nucleotides of any length, including but not limited to ribonucleotides or deoxyribonucleotides. No distinction in length is intended between these terms. Moreover, these terms refer only to the primary structure of the molecule. Thus, in certain embodiments, these terms can include triple-stranded, double-stranded and single-stranded DNA, PNA, and triple-stranded, double-stranded and single-stranded RNA. They also include modifications such as methylation and / or capping, as well as unmodified forms of polynucleotides.More specifically, the terms "nucleic acid", "polynucleotide" and "oligonucleotide" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers that contain non-nucleotide backbones, such as polyamide (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, provided that they contain nucleobases in a configuration that allows for base pairing and base stacking as found in DNA and RNA.

[0036] A "protein" as used herein is a biological molecule that is composed of a chain of one or more amino acids. Proteins differ from each other primarily in the sequence of amino acids dictated by the nucleotide sequence of the encoding gene. A peptide is a single linear polymer of two or more amino acids that are linked by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues; if there is more than one peptide in a chain, it may be called a polypeptide. A protein is composed of one or more polypeptides. Immediately after or even during synthesis, the residues of a protein are often chemically modified by post-translational modifications, which alter the physical and chemical properties, folding, stability, activity, and ultimately the function of the protein. Proteins sometimes have non-peptide groups attached, which may be called prosthetic groups or cofactors.

[0037] A "biological sample" as used herein includes any sample of material containing nucleic acids and / or proteins that can be extracted, analyzed and detected. Preferably, the material is in liquid or gas form, or can be dissolved or suspended in a liquid or gas, or can be liquefied or put into gas form, or otherwise prepared for analysis by the device and method of the present invention. Solid samples such as feces or soil samples can be loaded onto the chip, for example, in water. Aerosol biological samples can be used. A "biological sample" can be or be part of a tissue sample, a biological fluid sample, an environmental sample or other type of sample. Preferably, the biological sample is from a biological fluid, such as, but not limited to, blood, saliva, semen, urine, amniotic fluid, cerebrospinal fluid, synovial fluid, vitreous fluid, gastric fluid, nasopharyngeal aspirate and / or lymphatic fluid. A biological sample can also be a material or fluid contaminated with a source of nucleic acids and / or proteins. A biological sample can be a tissue sample, a water sample, an air sample, a food sample or a crop sample. Preferably, the biological sample analysis detects any one or more of water borne pathogens, air borne pathogens, food borne pathogens or crop borne pathogens.

[0038] The phrase "biological analysis" as used herein refers to the performance of biochemical assays that use cells, nucleic acids, proteins, or other biomolecules as starting materials and extract information for purposes of diagnosis, species identification, differentiation of point mutations and / or disease susceptibility alleles, qualitative analysis, quantitative analysis (e.g., to ascertain viral load), and other purposes as taught herein. One example of the application of this technology is pathogen detection, where genetic information is extracted from cells. The "steps" of "biological analysis" as used herein refer to the steps of extraction, optionally amplification, and detection.

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

[0040] As used herein, a "therapeutic treatment" is a planned treatment of a health problem.

[0041] As used herein, a "genetic analyzer" refers to a processor, such as a hardware computer processor or other dedicated digital signal processor, that receives electrical signals, stores electrical signals, outputs electrical signals, and processes the electrical signals, e.g., based on a computer program, where the electrical signals are related to a genetic analysis. For example, the genetic analyzer can be programmed to determine biomarkers based on input signals related to detected nucleic acids, and to determine and output an individualized genomic profile based on the biomarkers.

[0042] As used herein, a "dispensing controller" refers to a processor, such as a hardware computer processor or other dedicated digital signal processor, that receives electrical signals, stores electrical signals, outputs electrical signals, and processes the electrical signals, e.g., based on a computer program, where the electrical signals are associated with dispensing at least a portion of a product. For example, the dispensing controller can be configured to determine at least a portion of a product to dispense based, at least in part, on an individualized genomic profile.

[0043] As used herein, a "dispensing activator" refers to a device or system configured to dispense at least a portion of a product. For example, a dispensing activator may comprise at least a portion of one of: a vending machine; an automated teller machine; and a kiosk; and may comprise a three-dimensional printer configured to print at least a portion of a product or dispense a pre-packaged product.

[0044] Portions of the embodiments of the present invention, such as the genetic analysis portion, may be implemented using one or more computer systems, e.g., running on a computer processor and may be part of a program code. For example, the embodiments may be implemented using hardware, software, or a combination thereof. If implemented in software, the software code, whether provided to a single computer or distributed among multiple computers, may be stored on any type of non-transitory computer readable medium and loaded and executed on any suitable processor or collection of processors.

[0045] General Description As one skilled in the art will appreciate, the present invention may include hardware parts, software parts, and / or a combination of software and hardware parts. In one embodiment, the present invention is a portable processing means that interfaces with a compact integrated chip, which in one embodiment extracts, amplifies, and detects nucleic acids from a biological sample according to computer usable instructions embodied in a computer readable medium, and in another embodiment separates proteins, for example by electrophoresis. The system may use replaceable modules, as further described below in connection with FIG. 2.

[0046] The term "compact" as used herein refers to a microfluidic device layout design that minimizes the space used by fluidic channels, chambers, wells, and ports, as achieved by, for example, a microfluidic device layout, as discussed below. In one embodiment, the use of intersecting fluidic channels and shared wells is an example of a compact design. The term "integrated" as used herein refers to a microfluidic device layout design in which fluidic channels, chambers, wells, and ports used for various purposes are assembled on / within the same microfluidic device. For example, the embodiment of the chip shown in FIG. 4 includes 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 the chambers and channels mentioned in the previous sentence are specified in the plural, 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. The term "portable" as used herein refers to a system or device that can be easily carried or transported by hand or a mobile device. As used herein, the phrase "mobile device" refers to a small handheld device that typically has a display screen with touch input and / or a small keyboard and weighs less than about 10 kg, such as a smartphone, tablet, laptop or other portable medical device.

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

[0048] The processor means may comprise, for example, a personal computer, microprocessor or other suitable computer, such as a minicomputer, microcomputer, UNIX machine, one having an Intel processor or similar device, or a smart phone. It will also typically comprise conventional computer elements (not shown), such as a motherboard, a central processing unit (CPU), random access memory (RAM), disk drives, and peripherals such as a keyboard and a display. The RAM stores an operating system, such as Windows CE or other operating system, and appropriate software for processing signals relating to detected nucleic acids or proteins.

[0049] Portions of the embodiments of the invention described herein may be implemented using one or more computer systems. For example, the embodiments may be implemented using hardware, software, or a combination thereof. If implemented in software, the software code, whether provided to a single computer or distributed among multiple computers, may be stored on any form of non-transitory computer readable medium and loaded and executed on any suitable processor or collection of processors.

[0050] Further, it should be understood that the computer may be integrated into any of a number of forms, such as a laptop computer, a tablet computer, or a computer integrated into devices not normally considered computers but having suitable processing capabilities, including a personal digital assistant (PDA), smartphone, or any other suitable portable or mobile electronic device.

[0051] A computer may also have one or more input and output devices. These devices may be used, among other things, to present a user interface. Examples of output devices that may be used to provide a user interface include a printer or display screen for visual display of output, and a speaker or other sound generating device for audible display of output. Examples of input devices that may be used for a user interface include keyboards and pointing devices such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information via speech recognition or via another visual form.

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

[0053] Also, the various methods or processes described herein may be coded as software executable on one or more processors using any one of a variety of operating systems or platforms. Further, such software may be written using any of a number of 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 invention may be embodied as a computer readable medium (or multiple computer readable media) (e.g., a computer memory, one or more floppy disks, compact disks, optical disks, magnetic tapes, flash memory, field programmable gate array circuitry or other semiconductor devices, or other tangible computer storage media) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods for implementing the various embodiments of the invention described above. The computer readable medium or media are transportable such that the program or programs stored therein can be loaded onto one or more different computers or other processors to implement the various aspects of the invention described above.

[0055] In this regard, it should be understood that an implementation of the above embodiments includes at least one computer-readable medium encoding a computer program (e.g., a plurality of instructions) that, when executed on a processor, performs some or all of the above-described functions of the embodiments. As used herein, "computer-readable medium" encompasses only non-transitory computer-readable media that can be considered to be a machine or manufacture (i.e., an article of manufacture). A computer-readable medium can 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-transitory medium on which computer-readable information can be coded or stored. Other non-exhaustive examples of computer-readable media 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 media that can be considered to be a machine or manufacture.

[0056] As used herein, the terms "program" or "software" are used in a general sense to refer to any type 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 invention described above. Furthermore, according to one aspect of this embodiment, it should be understood that one or more computer programs that, when executed, implement the methods of the invention need not reside on a single computer or processor, but may be distributed in a modular manner among several different computers or processors to implement various aspects of the invention.

[0057] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, elements, and data structures that perform particular tasks or implement particular abstract data types. Typically the functionality of the program modules may 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. A 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 loads a sample 22 onto an integrated chip 20 (described in more detail below) and inserts the integrated chip 20 into a slot 16 on the system itself.

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

[0060] The phrase "modulated data signal" refers to a propagated signal that has one or more characteristics set or changed to encode information in the signal. Examples of modulated data signals include carrier waves or other transport mechanisms. Transmission media include any information delivery media. By way of example, and not limitation, transmission media include: wired media, such as a wired network or direct-wired connection, and wireless media, such as acoustic, infrared, radiometric, microwave, spread spectrum and other wireless media technologies.

[0061] The present invention may include a genome or transcriptome database that stores 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 a distant genome or transcriptome database, such as those stored at the National Institutes of Health, the Center for Disease Control, etc. Such a connection may facilitate tracking and adjusting responses to disease outbreaks at widely distributed analysis sites. Examples of analysis sites include hospitals, border crossings, airports, refugee camps, farms, quarantine areas, disease sites, homeless shelters, nursing homes, meat packing plants, and food processing centers. Those skilled in the art will recognize still other analysis sites to which the present invention is applicable.

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

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

[0064] In some versions of the invention, the device is mobile or POC and may be handheld.

[0065] Extraction-amplification-detection module The portable assay system 10 (FIG. 1) uses a combination of modules for extraction, amplification and detection of nucleic acids from a sample 22 (FIG. 1). The overall processing time, including any data processing 70 of the detected signals, is typically less than 20 minutes, and can 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 FIG.

[0066] As shown in FIG. 2, each of the modules may include one of a variety of implementations and combinations.

[0067] A biological sample 22 (FIG. 1) is loaded onto the integrated chip 20 (FIGS. 1 and 4). Loading of the biological sample may be accomplished manually through a sample inlet port 100 (FIG. 4) or through an automated loading means within the portable system 10. For example, the inlet port 100 may be loaded with a manual or automated loading device such as a pipette. Alternatively, in embodiments intended for field use, the inlet port 100 may be loaded directly with a cotton swab or a pricked finger by pressing the pricked finger against the inlet port 100. Capillary action causes blood to flow from the hole into the inlet port 100.

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

[0069] In one embodiment, the magnetic beads may have nucleic acid probes attached, optionally covalently. In this embodiment, a small electromagnet or magnet may be used to control the magnetic beads in the extraction module. The magnetic beads that may be used are any of the commercially available magnetic bead nucleic acid purification kits available from vendors 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] 2A, the extraction control module 40 can include a controller 250, a power source 252, and an electromagnet 254. Upon command from the extraction control module 40, the controller 250 applies power from the power source 252 to the electromagnet 254, which applies a magnetic field (not shown) to the extraction module 25. This causes the magnetic beads (not shown) in the extraction module 25 to cluster along the inner wall of the extraction chamber (not shown) of the extraction module 25. The controller 250 releases the magnetic beads by deactivating the power source 252, which causes the electromagnet 254 to cease applying the magnetic field.

[0071] The extracted nucleic acid may be amplified in the nucleic acid amplification module 27 (FIG. 4) of the integrated chip 20 under the control of the nucleic acid amplification control module 50 (FIGS. 2 and 3). Amplification may be accomplished using any suitable amplification technique, including conventional PCR techniques as disclosed in U.S. Patent Nos. 4,683,202 and 4,683,195, both of which are incorporated herein by reference in their entireties. Nucleic acids may also be amplified using isothermal techniques, such as those taught in U.S. Patent No. 7,494,791, which is incorporated herein by reference in its entirety.

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

[0073] Referring to FIG. 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 a nucleic acid template). The amplification may be proportional or exponential. In one embodiment, the module 50 includes a means for well-based nucleic acid amplification. As shown in FIG. 2B, such means may include a 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 Pelletier devices (WO 1998 / 9850147, which is incorporated herein by reference in its entirety) and thin-film-based devices. In other embodiments, the means for heating the nucleic acid amplification module of the integrated chip (see FIG. 4, element 25) may include infrared heating means (WO 1996 / 9641864, which is incorporated herein by reference in its entirety) or microwave radiation heating means.

[0074] 2B, the heating means 201 may include a heating / cooling element 203, a temperature sensor 205, a processor 207, a power control 209, and a power supply 211. Upon command from the amplification control module 50, the processor 207 initiates heating / cooling by directing the power control 209 to provide power from the power supply 211 to the heating / cooling element 203. The processor 207 monitors the temperature of the heating / control element 203 by means of the temperature sensor 205, and adjusts the command to the power control 209 as necessary to maintain the desired temperature for the amplification module 27.

[0075] In other embodiments, module 50 (FIGS. 2 and 3) includes means for fluid-based nucleic acid amplification (see, e.g., U.S. Pat. No. 7,041,481, which is incorporated herein by reference in its entirety). Such means may include a fluid flow generating means for generating a flow of buffer through the nucleic acid amplification module of the integrated chip, said fluid flow generating means being operatively coupled to a 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, e.g., U.S. Patent No. 7,315,376, the entirety of which is incorporated herein by reference).

[0077] In another embodiment, module 50 (see Figures 2 and 3) comprises a means for applying controlled tension to the 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 phrase "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 non-thermally driven processes of nucleic acid amplification or denaturation, or alternatives to annealing or primer extension. The application of "tension" to a nucleic acid is the result of applying a physical force to a nucleic acid strand other than that which is merely derived from thermal energy. Tension can be "precisely controlled" (as defined elsewhere herein) and / or can be adjustably controlled and / or variable.

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

[0080] The embodiment using the means for controlled tension on the nucleic acid strands in the nucleic acid amplification module of the integrated chip (see FIG. 4, element 25) has important advantages over the conventional (thermocycling method) means for nucleic acid amplification. These advantages include an overall better accuracy, as well as the length of the amplified sequence, the reaction yield and the reaction rate (total time of the amplification reaction), especially when amplifying "difficult" sequences (e.g., GC-rich sequences). Other important advantages include higher amplification efficiency and the ability to improve the fidelity of the amplification, for example by inducing proofreading exonucleases through the use of tension (see the teachings of U.S. Pat. No. 7,494,791, U.S. Pat. No. 8,632,973 and U.S. Patent Application No. 14 / 106,399, the teachings of all of which are incorporated herein by reference in their entirety).

[0081] An embodiment using a means for applying a controlled tension to a nucleic acid strand includes a mechanism for applying a variable and controlled amount of tension to a nucleic acid molecule held in a nucleic acid amplification module (see FIG. 4, element 25) of an integrated chip. Such a mechanism may comprise a first surface and a second surface having a means for immobilizing a nucleic acid molecule thereon, where the first and second surfaces are configured to move relative to one another. Alternatively, such a mechanism may comprise at least one surface having a means for immobilizing a nucleic acid molecule thereon, the device further comprising a mechanism for providing a controlled and variable fluid flow over the nucleic acid molecule. In yet another embodiment, such a mechanism may comprise at least one surface having a means for immobilizing a nucleic acid molecule thereon, further comprising a fluid flow passage distributed between the means for immobilizing the nucleic acid molecule. In other embodiments, such a mechanism may comprise a mechanism for providing a controlled and variable fluid flow over the nucleic acid molecule, configured to create a velocity gradient in the laminar fluid flow. Sometimes, such a mechanism may comprise a fluid flow channel, a stagnation point in the fluid flow, a counter-propagating fluid flow, or a combination thereof, configured to provide a velocity gradient in the laminar fluid flow. In other embodiments, such mechanisms 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 holding nucleic acids within the nucleic acid amplification module of the integrated chip (see FIG. 4, element 25) may comprise activatable primers that include conjugation groups for immobilizing extension products obtained during nucleic acid amplification. Alternatively, nucleic acid polymerases may be immobilized or otherwise held on the surface of the integrated chip 20.

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

[0083] In various embodiments, the module 60 may comprise a fluorescent or electro-optical detection means 950 for detecting nucleic acids, as shown in FIG. 9A. The fluorescent detection means 950 may include a light emitter 952, such as a light emitting diode and / or a laser diode, a data acquisition device 954, such as a photodetector, a photomultiplier tube (PMT) or a 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 the sample with emitted radiation 960, producing a fluorescent signal 962 that is sensed by the data acquisition device 954. In the embodiment shown in FIG. 9A, the emitted radiation 960 illuminates the detection module 29 in a transmission geometry, although it should be understood that reflection and other geometries may also be used. The data acquisition device 954 converts the fluorescent signal 962 into a conditioned data signal 958 that is detected and recorded by the processing unit 956.

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

[0085] 4, in one embodiment, CE is performed in a detection channel 112, which may be filled with a suitable buffer and may be pre-filled with a sieving matrix such as agarose, hydroxypropylcellulose or polyacrylamide, among others. In certain embodiments, an end user may fill the detection channel 112 (FIG. 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 Affymetrix, Santa Clara, CA.

[0087] In one embodiment, the nucleic acid detection control module 60 comprises an electrochemical means for detecting nucleic acids. An example of an electrochemical means for detecting nucleic acids is provided in US Patent Application Publication No. 2008 / 0081329, which is incorporated herein by reference in its entirety. Briefly, such means implements 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 bound to the conductive surface and capable of binding to the target substrate. The probe bound to the conductive surface is contacted with the test sample to form a surface-bound target complex. The surface-bound target complex further includes a first redox complex. The surface-bound probe or the surface-bound target complex, if present, is contacted with a fluid medium containing a second redox complex, where one of the first and second redox complexes is a redox transition metal complex capable of performing 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 reduced transition metal complex. The redox-transition metal complex does not undergo any significant amount of redox reaction in the absence of the redox catalyst complex. The redox reaction of the redox-transition metal complex catalyzed by the redox catalyst complex is detected.

[0088] In another embodiment, the nucleic acid detection control module 60 includes an impedance measuring means for detecting a nucleic acid. An example of an impedance measuring means for detecting a nucleic acid is described, for example, in U.S. Patent No. 7,169,556, which is incorporated herein by reference in its entirety. Briefly, the method includes contacting a nucleic acid having a first portion and a second portion with a substrate having oligonucleotides attached thereto, the oligonucleotides located between a pair of electrodes having a sequence complementary to a first portion of the sequence of the nucleic acid, the contacting occurring under conditions effective to allow hybridization of the nucleic acid with the oligonucleotides on the substrate. After hybridization of the nucleic acid and the oligonucleotides has occurred, the substrate-bound nucleic acid is contacted with a first type of conductive particles (e.g., metal beads), the conductive particles being made of a material capable of conducting electricity and having one or more types of oligonucleotides attached thereto, at least one of the types of oligonucleotides having a sequence complementary to a second portion of the sequence of the nucleic acid, the contacting occurring under conditions effective to allow hybridization of the nucleic acid with the oligonucleotides on the substrate to form a conductive particle-complexed test substrate. After the second hybridization has occurred, the test substrate is contacted with a salt solution at a salt concentration effective to sufficiently dehybridize and remove non-specifically 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 an embodiment of the present invention, any one of many possible techniques for extraction, amplification and detection may be used in combination; or indeed, one or more of extraction, amplification and detection may be omitted altogether from the combination of extraction, amplification and detection. For example, in the modular system of FIG. 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(s) 40 may be used in combination with any one of the possible types of modules shown under amplification module 50 or with another type of amplification module; further, such extraction module(s) 40 and amplification module(s) 50 may be used in combination with any one of the possible types of modules shown under detection module 60 or with another type of detection module. Depending on the target to be detected, different combinations of such modules may be used. First, a biological sample such as blood, saliva, etc. may be used as the biological sample to be analyzed; or a solid sample (e.g., a fecal sample) or an aerosol sample may be used. In the extraction module 40, the target nucleic acid or protein is separated from the remaining biological sample. Amplification can then occur in 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 any of the other detection modules shown in Figure 2, or other types of detection modules including, for example, a melting curve analysis module, a chemiluminescence module, a quantum dot module, a module that uses nanoparticles such as gold nanoparticles, or a detection module based on the use of radiation. In another example, if the initial biological sample to be analyzed is a 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.Regarding the amplification module 50, in addition to the type of modules shown in Fig. 2, other possible modules that can be used are Loop Mediated Isothermal Amplification (LAMP) module, Helicase-Dependent Amplification (HDA) module, SDA (Strand Displacement Amplification) module or Bridge Amplification module. LAMP technology such as that described in T. Notomi, et al., Nucleic Acids Research, 28, e63 (2000) can be used; HDA technology such as that described in Vincent M, Xu Y, Kong H. (2004), "Helicase-dependent isothermal DNA amplification," EMBO Rep 5 (8): 795-800 can be used; SDA technology such as that described in GT Walker, et. al., Proc. Natl. Acad. Sci USA, 89, 392-396 (1992) can be used; all of these references are incorporated herein in their entirety by reference. The modules of the system may be interchangeable between thermal and isothermal amplification modules; any of the amplification techniques taught herein, such as Helicase-Dependent Amplification (HDA), Strand Displacement Amplification (SDA), and 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 to Nanobiosym, Inc., the entire teachings of which are incorporated herein by reference. In one specific embodiment, the portable assay system 10 includes an extraction control module 40 configured to use magnetic beads for extraction of nucleic acids, an amplification control module 50 that uses a thermal cycling method using a Peltier device, and a detection control module 60 that separates and detects nucleic acids using capillary electrophoresis and fluorescent signal detection.

[0090] According to an embodiment of the present invention, different integrated chips may be capable of being analyzed by a single mobile device, each possible different integrated chip implementing a different functionality that the single mobile device recognizes and executes, for example, one chip may be used for the analysis of nucleic acids and another chip may be used in the same mobile device for the analysis of proteins.

[0091] A diagram of the general flow of materials and data through a system of the invention, such as the modular system depicted in FIG. 2, is shown in FIG. 3. The extraction, amplification and detection modules 40, 50, 60 typically operate in about 4 minutes each, compared to hours to weeks using conventional techniques. In embodiments according to the 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 the analysis of the sample. In one embodiment, the integrated chip 20 housing the extraction, amplification and detection modules 40, 50, 60 typically weighs about 35 g, while the application specific integrated circuit (ASIC) used to process the detection signal weighs about 25 g, making the entire cartridge weigh about 60 g. The modules are located on the integrated chip and are controlled by a corresponding control module (not shown) of the portable assay system 10.

[0092] Similarly, in some embodiments, a Peltier device or thin film heater or other temperature control means may be used to heat and cool the nucleic acid amplification chamber 108 (FIG. 4), while a thermocouple monitors the temperature of the chamber 108. The capillary electrophoresis process may be controlled by a detection control module, which controls the strength and duration of the electric field applied across the channels used for capillary electrophoresis.

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

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

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

[0096] In certain embodiments, signal intensity versus time may be analyzed to provide an indication of the type and amount of nucleic acid species present in the biological sample 22 (FIG. 1). For example, by plotting the detected signal against the signal produced by a reference sample, a user may be able to determine whether a pathogen present in the reference sample is present in the test sample, since identical pathogens will produce signal peaks at the same points on the time axis (assuming identical analytical conditions). The data processing means further includes a viral load calculation means for calculating the viral load (e.g., by integrating the area under the curve representing the fluorescent signal as a function of time).

[0097] In another embodiment, the system 10 further comprises a data display means for outputting the data.

[0098] The portable assay system 10 interfaces with the integrated chip 20 through electrical, chemical and mechanical interfaces. Clamps and / or fasteners secure the chip in place sufficiently stable to prevent vibration from displacing the location of the electrodes, detectors (such as photodetectors) or heaters. Heaters (and optionally cooling elements such as inkjet coolers) (not shown) located below, above or on the amplification control module 40 heat and / or cool specific locations of the chip, as described below. Similarly, electrodes located near the detection control module 60 may be used to control separation and fluid flow through the chip, as described below. Because the chip 20 is transparent, the wells, chambers and channels of the chip 20 may be probed using electromagnetic beams, such as infrared or electromagnetic beams for heating, or optical beams for fluorescent tag identification.

[0099] Integrated Chip While the following description refers to a specific embodiment of integrated chip 20 (FIGS. 1 and 4), it is understood that other chip designs may be used with the portable assembly system 10 (FIG. 1) of the present invention.

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

[0101] The integrated chip 20 may be formed from glass, any plastic having good optical properties including, 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.

[0102] The integrated chip comprises 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 biological samples, the extraction chamber being 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, the nucleic acid amplification chamber being 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, the detection channel being connected to the nucleic acid amplification chamber by at least one amplification product transport channel. Preferably, the at least one sample inlet port, the at least one extraction chamber, the at least one amplification chamber and the at least one detection channel are arranged to minimize the use of space. As discussed above in relation to FIG. 2, replaceable modules may be used.

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

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

[0105] The integrated chip may further comprise at least two reagent addition ports, each in fluid communication with each nucleic acid transport channel; at least two sample wells, each in fluid communication with each amplification product transport channel. Preferably, the at least two sample wells are configured for use in the addition and / or disposal of additional 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, the at least one sample waste well is configured for use in adding and / or disposing of additional reagents.

[0107] In other embodiments, the integrated chip further comprises at least one buffer waste well in fluid communication with the at least two detection channels; and a buffer well in fluid communication with the at least two detection channels. Preferably, the at least one buffer waste well and the buffer well are configured for use in the addition and / or disposal of additional reagents.

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

[0109] Preferably, each sample waste channel is coupled to a detection channel downstream of at least one amplification product transport channel, thereby effectively increasing the cross-section of the at least one amplification product transport channel and the 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 comprises microfabricated protrusions (posts) disposed on at least one nucleic acid extraction chamber and / or at least one sample transport channel. As described above, the microfabricated protrusions disposed on the integrated chip can be used for mechanical shearing of cells of a biological sample, for example, as described in U.S. Patent No. 5,635,358 and WO 2006 / 06029387, the entireties of which are incorporated herein by reference.

[0111] The integrated chip may further comprise at least two electrodes for applying a voltage across at least one detection 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 aspect, the invention is a novel valve assembly, as shown in Figure 5 and Figures 6A and 6B and described in further detail below. Thus, in one embodiment, the invention is a valve assembly in a microfluidic device.

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

[0114] In another embodiment, the valve assembly comprises a transport channel for fluid transport; a ferrofluid channel intersecting the transport channel; a first ferrofluid reservoir and a second ferrofluid reservoir, both connected by a ferrofluid channel; a ferrofluid in either one or both of the ferrofluid reservoirs; and a permanent magnet or electromagnet for filling the ferrofluid channel with the ferrofluid. Ferrofluids are typically colloidal mixtures that contain magnetic particles suspended in a liquid, with additional detergents / surfactants mixed into the liquid to prevent the particles from clumping 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 ferrofluid may be used, such as, for example, ferrofluids available from Ferrotec Corporation, Bedford, NH.

[0115] In yet another embodiment, and with reference to Figures 7A and 7B, the present invention provides a valve assembly in a microfluidic device comprising: a reservoir; an inflow transport channel for transporting a fluid having an output end connected to the reservoir; first and second outflow channels for transporting a fluid, each having an input end connected to the reservoir; a magnetic fluid disposed in the reservoir; and a magnet for controlling the position of the magnetic fluid in the reservoir. During operation of the valve assembly of Figures 7A and 7B, the magnetic fluid in the reservoir closes either the output end of the inflow channel; or the input end of the first outflow channel but not the input end of the second outflow channel; or the input end of the second channel but not the input end of the first outflow channel; or is disposed such that neither the output end of the inflow channel nor the input ends of the first and second outflow channels are closed.

[0116] In the embodiment shown in FIG. 4, the integrated chip 20 includes eight sample regions 130, each including 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 is 1-4 inches long and 1-4 inches wide; preferably, the integrated chip 20 is approximately 2 inches wide and 4 inches long.

[0117] The nuclear sample area 130 includes one sample inlet port 100, one extraction chamber 104, one amplification chamber 108 and one detection channel 112. It is understood that for each biological sample, there is a separate and identifiable path from each sample inlet port to each extraction chamber, 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 (e.g., sample waste well 118 and buffer well 112) that are shared by multiple sample areas. This sharing reduces the space required for the integrated chip layout and also reduces material waste.

[0118] It is understood that a chip may be configured with any number of sample regions (e.g., 1, 12, 24, 256, 386, or 512), provided the size of sample region 130 is sufficient to extract, amplify, and detect nucleic acids. Given the appropriate referencing available, sample region 130 may be used to analyze multiple different samples for the same target biomarker sequence or pathogen pair, one sample for multiple different pathogens or biomarkers, or any suitable order or combination of samples and pathogens or biomarkers.

[0119] The biological sample 22 (see FIG. 1), reagents used for extraction, amplification and detection, travel between the different wells and reaction chambers through a microfluidic transport channel 102, as shown in FIG. 4. In a specific embodiment, the transport channel 102 has a cross-sectional area of ​​10-1000 μm×10-1000 μm, preferably 200×100 μm, and is large enough to accommodate the flow of magnetic beads used in the extraction process embodiment. In one embodiment, the ports and wells, such as the sample inlet port 100, the reagent inlet / outlet port 108, the buffer waste well 114, the buffer well 116, the sample waste well 118 and the sample well 120, are 0.5-10.0 mm in diameter, preferably 0.9-5.0 mm in diameter, with the depth determined by the thickness of the integrated chip 20. The nucleic acid amplification chamber 108 is typically 0.1-30.0 mm in diameter, preferably 10.0 mm in diameter, and 10 μm-19.0 mm in depth, preferably 0.5 mm in depth. The nucleic acid extraction chamber 104 may be 1 to 25 mm long and 1 to 30 mm wide, preferably 15 mm long and 10 mm wide. 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] Fluid flow through the channels, ports, wells and chambers is controlled by a combination of valves and propulsion means, which can be a syringe of a manual pump connected to the sample inlet port 100. Other propulsion means such as peristaltic and piston pumps can also be used. The propulsion means pushes the fluid through the channels and chambers unless a closed valve blocks the fluid flow. The valves can use any one of several technologies, including manually actuated plugs, mechanically actuated plugs, electromagnetically actuated plugs, ferrofluidic valves, pneumatic valves or any other suitable technology.

[0121] 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 may 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 may be inserted manually or may be mechanically actuated. In one embodiment, the inlet sample well 100, the reagent inlet / outlet port 106, the sample waste well 118, the sample well 120, the buffer waste well 114, and the buffer well 116 may act as valve ports 151 to receive the passive plug valve 150.

[0122] 6A and 6B show an alternative method of controlling fluid flow using a ferrofluid valve 600. Ferrofluids are typically colloidal mixtures that contain magnetic particles suspended in a liquid, with additional detergents / surfactants mixed into the liquid to prevent the particles from clumping together (see also Berger, et al. (July 1999). "Preparation and properties of an aqueous ferrofluid". Journal of Chemical Education 76 (7): pp. 943-948, incorporated herein by reference in its entirety). Any commercially available ferrofluid may be used, such as, for example, ferrofluids available from Ferrotec Corporation, Bedford, NH. The in-line ferrofluid valve 600 comprises a ferrofluid channel 601 disposed 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 dirt reservoir 606. In the "open" state, the magnetic fluid 602 remains within the magnetic fluid reservoir 604, allowing the fluid to flow through the transport channel 102 in a fluid flow direction 608, as shown in Figure 6A. By placing a magnet 610 near the dirt reservoir 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 show how a 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 embodiment shown in 7A and 7B, fluid enters a well 702 via an inlet 704. A small amount of magnetic fluid 602 remains in the well 702. With both outlets 706a and 706b in the open position, as shown in Figure 7A, the magnetic fluid 602 rests in a stable position inside the well 702. 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 can have multiple inlet and outlet ports 704, 706.

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

[0125] Referring again to the embodiment shown in FIG. 4, in one embodiment of the present invention, a 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 can be moved along the microfluidic transport channel 102 to the nucleic acid extraction chamber 104 under pressure of a syringe pump or other suitable fluid pressure generating means. Alternatively, the magnetic beads can be already loaded into the extraction chamber 104. Next, a user 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 through the same transport channel 102 from the sample inlet port 100 to the extraction chamber 104. The magnetic beads (not shown) are attached to the nucleic acids of the biological sample 22 by the streptavidin coating. Once the attachment process is complete, a magnetic field is applied to the extraction chamber, causing the magnetic beads (and the attached nucleic acids) to move in the direction of the magnetic field, extracting the nucleic acids from the biological sample 22.

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

[0127] Once the extracted nucleic acids are free of the mixture, they are forced downstream (e.g., by applying pressure with a syringe or pump) into the nucleic acid amplification chamber 108, which the user then loads with reagents via the sample inlet / outlet port 106. The user then isolates the amplification chamber by closing appropriate valves before initiating amplification by conventional amplification techniques. A thermoelectric heater (not shown) may be used to heat and cool the reagents in the amplification chamber 108. In one embodiment, the reagents include primers, a DNA polymerase (such as Taq polymerase), deoxynucleotide triphosphates, a buffer solution, and divalent cations.

[0128] Once the nucleic acid has been sufficiently amplified, the valve isolating the amplification chamber 108 is opened, allowing the amplified nucleic acid to flow through the delivery channel 102 into a sample well 120 which is 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 biological sample 22. Capillary electrophoresis involves passing an electric current through an electrolyte, such as an aqueous buffer solution mixed with a test sample in a short (approximately 50 μm long) channel, such as detection channel 112. The electric current causes the sample to move downstream through the detection channel; however, the compounds separate as they migrate, because the migration speed of the compounds depends on their molecular weight, the current, and the size of the channel, among other variables.

[0130] In one embodiment, the user may add electrolyte and sieving matrix to the detection channel 112 through the buffer well 116 (also shown in FIG. 4). In an embodiment where the buffer well 116 is connected to multiple detection channels 116 as shown in FIG. 4, simultaneous loading of all detection channels is achieved. Once the buffer well 116 is filled, an electric field 130 applied to electrodes located 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 118 through the detection channel 112 as shown in FIG. 8B. Typically, the electric field 130 is about 400V. The extracted nucleic acid accumulates in an accumulation area 113 located in the detection channel 112 between the intersection points of the transport channel 102 connecting the sample well 120 and the sample waste well 118 to the detection channel 112. Waste from the sample well 120 continues to the sample waste well 118.

[0131] 8C shows how application of an electric field 131 across the detection channel 112 from the buffer well 116 to the buffer waste well 114 causes separation of the amplified nucleic acid 23 sample into species 140 according to molecular weight. Typically, the strong electric field 131 is about 6 kV. Because different target biomarker sequences or amplicons of different pathogens have different molecular weights, the different biomarkers or pathogens migrate downstream in the detection channel 112 at different velocities. An unknown species can be identified by comparing its velocity to that of a known reference (not shown) of the same detection channel 114 or the same detection 112 under the same strength of electric field 131.

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

[0133] In one embodiment, the fluorescent signal 1010 is produced by illuminating the isolated load 140 with a laser beam 1004 or an LED or other light source of suitable frequency. A laser 1002 produces a laser beam 1004 which may be directed to the measurement area using a mirror 1006. Alternatively, a lens may be used to focus the laser beam 1004 on the sample. The beam may also be produced by a light emitting diode or other light source instead of a laser, and may or may not be coupled via a fiber optic cable. It will be appreciated that a variety of optical arrangements may be used to interrogate the species 140.

[0134] The laser beam 1004 excites the separated load 140, producing a fluorescent beam 1010, which is split from the laser beam 1004 by a dichroic beam splitter 1007. In an 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 detection signal to a reference signal trace 1021 of a known reference sample in an output signal graph 1020, where matching peaks in the trace indicate the presence of the same biomarker sequence or pathogen. The areas under the peaks are integrated to provide an indication of the relative amount of biomarker or pathogen (i.e., viral load) present in each sample. In an embodiment, the software correlates the output signal graph 1020 with the reference signal trace 1021 using standard correlation techniques and provides an output suitable for interpretation by a user.

[0135] Lab-on-a-chip for nucleic acid or protein detection An embodiment of the present invention includes an integrated chip capable of performing the above biological assays in a single device, also called an integrated microfluidic chip or lab-on-a-chip. The integrated chip is designed to sequentially perform 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 an integrated microfluidic chip or lab-on-chip similar to that discussed in the current section can be used for protein detection, with the technology appropriately modified for use in protein detection. For example, in protein detection, in one embodiment, only an extraction module and a detection module without amplification can be used. Furthermore, in some embodiments, only a detection module can be used. In other embodiments, only an extraction and nucleic acid sequencing and detection module can be used without an amplification module. Any one of these modules can be used alone or in combination with each other.

[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 that includes microfluidic channels in serial fluid communication with functional modules: a nucleic acid extraction module, a nucleic acid amplification module and a nucleic acid separation module.

[0138] In one embodiment, a biological sample of a subject is loaded onto the integrated chip, and nucleic acids are extracted from the biological sample in an extraction module. For example, the biological sample includes cells, and cellular DNA is extracted from the cells. The extraction process preferably utilizes precipitation of nucleic acids using magnetic beads. The extracted nucleic acids are then transported by fluid pressure to a nucleic acid amplification module, where the nucleic acids are amplified using polymerase chain reaction. In some embodiments, the amplification utilizes thermal cycling. In other embodiments, the amplification utilizes isothermal techniques of amplification, including but not limited to the teachings of U.S. Patent No. 7,494,791, U.S. Patent No. 8,632,973, and U.S. Patent Application No. 14 / 106,399, all to Nanobiosym, Inc., all of which teachings are incorporated herein by reference. The amplified nucleic acid products are then transported by fluid pressure to a nucleic acid separation module, where the nucleic acids are separated and detected using capillary electrophoresis. Preferably, fluorescent nucleic acid primers are used to detect the nucleic acid products using fluorescent detection. 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 controlling, respectively: (1) application of a magnet for precipitation of the magnetic bead-attached nucleic acids, (2) thermal cycling during amplification of the extracted nucleic acids, and (3) fluorescence-assisted detection of the nucleic acids.

[0140] 10, the present invention, in various embodiments, enables advances in healthcare and other industries. The integrated chips and hardware systems of the present invention, described in detail below, can be used for analysis of biological samples in biomedical testing in biodefense, environmental testing, food borne 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 FIG. 4, FIG. 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 processes are achieved with closed chambers, open wells, and interconnecting channels, the combination of which constitutes the microfluidic chip. In this integrated chip 20, the multiplex design to accommodate the analysis of multiple samples is achieved by a structure in which each sample has an independent fluid path in separate sample regions 130 from input to final detection without any 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 (e.g., sample waste well 118 and buffer well 116) shared by the multiple sample regions 130. This sharing of a common well reduces the space required for integrated chip layout 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 chip, thus accommodating eight samples. These wells 100 are then each connected by a channel 102 to a set of closed extraction chambers 104 designated for nucleic acid extraction. These extraction chambers 104 are then each connected to another set of open wells (reagent inlet / outlet ports 106) allowing the flow-through of fluids through the extraction chambers 104 as required to perform the extraction assay. In a preferred embodiment, the flow of fluids 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 ports 106 then connect to another set of closed amplification chambers 108 designed for amplification. The amplification chambers 108 also connect to sample wells 120 on one side and to reagent inlet / outlet ports 106 (each shared with the extraction chamber 104) on the other side, again allowing fluid flow-through through the amplification chambers 108.

[0144] Finally, each of the fluid paths terminates in a set of independent and distinct separation / detection channels (CE channels) 112 towards the end of the CE channels 112 where electrophoretic separation is accomplished prior to fluorescence detection. The electrophoresis module is once again uniquely designed to multiplex the injection and separation of all eight samples that have already been subjected to extraction and amplification processing on-chip.

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

[0146] Typically, chip 20 is about the size of a thick credit card. In one embodiment, 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, integrated chip 20 is about 5 cm wide, 10 cm long, and 2 mm thick. Those skilled in the art will appreciate that the size and design of a particular chip is a matter of design preference.

[0147] In a preferred embodiment, the characteristic dimensions of the chip 20 are 10-200 μm×10-200 μm (e.g., 50 μm×20 μm) for the detection (CE) channel 112 and 10-1000 μm×10-1000 μm (e.g., 200 μm×100 μm) for all other channels. It will be understood that for the channels through which the magnetic beads flow, the dimensions of the channel are selected to ensure unobstructed flow of the magnetic beads. The detection (CE) channel 112 can be 10-120 mm (e.g., 80 mm) in length. A larger detection CE channel 112 size requires a larger voltage for CE analysis. The size of the transport channel 102 and other channels, such as the extraction chamber 104 and the reagent inlet / outlet port 106, the reagent inlet / outlet port 106 and the amplification chamber 108, the amplification chamber 108 and the sample well 120, and the channel connecting the sample well 120 and the detection channel 112, is not critical.

[0148] In a preferred embodiment, circular wells and chambers such as the buffer well 116 are 5-15 mm (e.g., 10 mm) in diameter and 0.05-1 mm (e.g., 0.5 mm) deep. Hexagonal chambers such as the extraction chamber 104 can be 5-25 (e.g., 15 mm) long (at both narrowed ends) and 5-15 mm (e.g., 10 mm) wide. This type of hexagonal design promotes easy fluid flow in the extraction chamber 104, especially since the process involves a large volume of fluid flow. Other embodiments may accommodate circular chambers with diameters of 0.1-30 mm and depths of 0.01-19 mm, and hexagonal chambers that are 1-25 mm long (at both narrowed ends) and 1-30 mm wide.

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

[0150] Fabrication of compact integrated chips The embodiments of the microfluidic chips made 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 aspect thus ensures that issues such as carryover contamination, a common problem with reusing biological processing tools, are minimized to the point of elimination. These chips can also be manufactured from glass, silicon, and other materials.

[0151] The fabrication process involved in the production of these chips involves microfabricating structures in silicon (Si) or glass or aluminum (Al) or other such materials to create a master with the desired microfluidic structures. This creates a negative microfluidic structure on the microfabricated Si / glass device. The process then involves casting a rubber-based mold on the microfabricated Si / glass device to form a positive mold. Hot embossing of the rubber mold on a plastic plaque creates the microfluidic structures identical to the original 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 follow the pattern of the rubber mold, creating negative structures in the plastic plaque identical to the negative structures of the original Si / glass microfabricated device. The plastic plaque with the negative structures embossed on it is finally pierced and glued 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, polystyrene, or other such materials may be used, depending on the customization required by a particular application.

[0153] The above process of hot embossing to fabricate plastic microfluidic chips allows for the production of low-cost devices suitable for disposable use. Furthermore, because plastic manufacturing is typically scalable with non-linear cost ratios, these microfluidic devices can be batch processed, greatly reducing the cost of manufacturing. The following biological assays can be performed using the fabricated plastic microfluidic chips:

[0154] Modular Design 4, the chip 20 includes three modules: a DNA extraction module 25, a PCR amplification module 27, and an electrophoretic separation module 29 involved in 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 fluid flow from one module to the next.

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

[0156] Nucleic Acid or Protein Extraction Module It will be appreciated that extraction modules similar to those discussed below in this section can be used for protein extraction, with the following nucleic acid techniques appropriately modified for use in protein extraction.

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

[0158] These beads can optionally be kept in the chamber for subsequent amplification directly from the DNA-attached beads or by eluting the DNA from the beads prior to amplification. To perform the extraction assay, 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 protocols well known in the art) is first mixed with the cell sample and the extraction process is carried out according to the following protocol. The components and specifications of the buffers and beads used in the extraction protocol are also detailed in the following protocol.

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

[0160] The magnetic beads / lysis solution is then loaded into the extraction chamber containing the previously dispensed cell sample via well 100. Dispensing the bead solution into the extraction chamber causes mixing of the cells and bead solution during flow.

[0161] The phrase "lysis buffer" as used herein refers to any buffer used to lyse cells for use in experiments to analyze cellular compounds. Typically, lysis buffers include detergents that cause cell membrane disruption. Examples of such detergents include Triton X-100, NP40, or Tween-20. Other components of the lysis buffer may include protease inhibitors, DNA enzymes; magnesium salts, lysozyme, disulfide reducing agents such as β-mercaptoethanol, ion chelating agents such as EDTA, and preservatives such as sodium azide. One of skill in the art will understand that the particular buffer used will be determined based on the starting material and the target to be analyzed.

[0162] The loading of the magnetic beads is stopped once the extraction chamber 104 is filled. The filled extraction chamber is incubated at room temperature for a few minutes to allow the target, e.g. DNA, to bind to the ligand coated on the magnetic beads.

[0163] In some embodiments, once incubation is complete, a magnet is placed (or engaged, if an electromagnet is used) beneath the extraction chamber 104 (FIG. 4) of the chip 20 below the extraction chamber 104. The magnet precipitates the magnetic beads, allowing them to be retained within the extraction chamber 104 as the cellular debris is washed away. Typically, the magnet used exerts a field of about 12,000-13,000 Gauss at the extraction chamber. It will be appreciated that the strength of the magnet will depend on the size of the beads, the size of the sample and other factors and may be adjusted accordingly.

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

[0165] In some embodiments, the washing step may be repeated a sufficient number of times to ensure that the purified target (e.g., DNA) is free of lysis solution and cellular debris. One skilled in the art would be able to easily ascertain the number of washing cycles required to achieve this. The captured target molecules (e.g., DNA) may be eluted in the extraction chamber 104 using standard elution buffers, as known in the art.

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

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

[0168] As used herein, the phrase "master mix" refers to a solution containing 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). One of skill in the art will understand that a particular master mix will be selected based on the target to be amplified, the test conditions, and the preferred polymerase.

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

[0170] If the nucleic acid-bound beads are to be used directly in the amplification, the master mix is ​​loaded into the amplification chamber 108. The magnet under the extraction chamber 104 is then disengaged, allowing 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 channels 102 connecting the inlet / outlet ports 106 and wells 120 to the extraction chamber 104 can be sealed using elastic or ferrofluidic valves. The beads are then free to move within the extraction chamber 104 but are prevented from leaving it. The extraction is complete and the chip is ready to be subjected to amplification.

[0171] Otherwise, if no DNA-bound beads are 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 removed and unloaded from the extraction chamber 104. The magnet below the extraction chamber 104 is then disengaged and the well 100 and the reagent inlet / outlet port 106 are sealed using appropriate valves.

[0172] The chip is then heated to 50-90°C (e.g., 70°C) on a Peltier device or other suitable heat source typically used as a means to control or regulate temperature, such as in temperature control methods for amplification. This causes the elution of DNA from the magnetic beads.

[0173] After heating, a valve opens blocking the transport of fluid from the extraction chamber 104 to the amplification chamber 108 through the transport channel 102. Finally, the extracted DNA / RNA / protein is flushed from the extraction chamber 104 to the amplification chamber 108.

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

[0175] Amplification Module According to embodiments of the invention, as discussed elsewhere herein, the amplification module may be isothermal or thermal. Similarly, according to other embodiments of the invention, as discussed elsewhere herein, the amplification module may be replaced by only one cycle of amplification or DNA / RNA replication or DNA / RNA sequencing, which process may be isothermal or thermal. In another embodiment, no amplification is performed.

[0176] In one embodiment, the extracted DNA / RNA from the extraction module and an amplification master mix are mixed in the amplification module 108. The amplification master mix contains (apart from other standard reagents described below) a set of sequence-specific forward and reverse primers that amplify a genetic sequence of interest (usually a species-specific signature) in the extracted DNA / RNA.

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

[0178] In some embodiments, amplification is accomplished by deploying an integrated chip with means for precise temperature control and, for example, programmed temperature cycling protocols (see, for example, the protocol for amplification of a 700 bp DNA fraction characterized in E. coli DH10B cells described in the Exemplification section). The number of cycles can be easily ascertained by one of skill in the art based on the starting concentration of the sample and the desired concentration of the final amplification product.

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

[0180] In one embodiment, the amplification is performed using hardware modules, which are described in detail below.

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

[0182] Completion of the heat or tension cycles leads to amplification of the DNA / RNA sequence, as designed by the primers, which is ready for analysis using the methods described below.

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

[0184] Referring again to FIG. 4, the detection (CE) channel 112 is loaded with a polyacrylamide sieving matrix (also referred to herein as a “gel”) (e.g., GenceScan polymer, ABI, or a homemade polyacrylamide solution suitable for use in capillary electrophoresis). This is accomplished by dispensing the sieving matrix from a central buffer well 116 on the integrated chip 20 to all eight multiplexed separation modules 29 simultaneously. Next, a 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., buffer is loaded into the CE buffer well 116, the CE buffer waste well 114, and the CE sample waste well 118, but not into the CE sample well 120). The CE sample well 120 is first loaded with the amplification product by forcing the sample from the amplification chamber 108 into the CE sample well 120 with pressure. The running buffer is then added to the amplification product in the CE sample well 120.

[0185] Those skilled in the art will also understand that different gel media are 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, a low concentration of acrylamide can be used to separate high molecular weight proteins (e.g., 5% for 40-200 kDa), while a high concentration of acrylamide can be used to separate low molecular weight proteins (e.g., 15% for 10-40 kDa). Similarly, a low concentration of agarose can be used to separate high molecular weight nucleic acids (e.g., 0.5% for 1-30 kbp), and a high concentration of agarose can be used to separate 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 an agarose matrix), Tris / glycine / SDS (e.g., for proteins separated on a polyacrylamide gel).Those skilled in the art will understand that the choice of running buffer depends on the target to be analyzed.

[0187] Alternatively, if size standards are also used to confirm the size of the amplified products, the size standards (e.g., Gene Scan TAMRA orr ABI) can be added to the amplified products and running buffer can be loaded on top. The running buffer and amplified DNA can be mixed to form a homogenous suspension in well 120. In this case, primers labeled with a fluorophore similar to the size standards (e.g., VIC, ABI) to allow fluorescent detection of DNA can lead to labeling of the amplified DNA as would be expected by the amplification process.

[0188] Any molecular weight standard suitable for nucleic acid separation can be used, examples being GelPilot® molecular weight markers available from QiaGen, 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, e.g., Prudnikov et. al. Nucleic Acids Res. 1996 November 15; 24(22): 4535-4542 or the protocols available online at the URL http: / / info.med.yale.edu / genetics / ward / tavi / n_label.html, the entire teachings of both of these publications are incorporated herein by reference). Examples of fluorescent labels include fluorescein, 6-carboxyfluorescein, 5'-tetrachloro-fluorescein, Texas Red (sulforhodamine 101 acid chloride), quantum dots, and fluorescently tagged nanoparticles.

[0190] 8A-8C illustrate the CE process carried out in the detection (CE) channel 112. Once the reagents are loaded into the wells 114, 116, 118 and 120, a platinum electrode grid (not shown) is lowered onto the chip 20 (FIG. 4) to immerse the ends of the electrodes into all of the wells 114, 116, 118 and 120. As shown in FIG. 8B, a short-term, relatively low injection voltage (e.g., about 400 V) electric field 130 is applied between the sample well 120 and the sample waste well 118. This causes the amplification product (amplified nucleic acid 23) to migrate along the portion of the transport channel 102 and detection (CE) channel 112 that connects the sample well 120 and the sample waste well 116. Next, as shown in FIG. 8C, a strong electric field 131, such as a high voltage of about 6 kV, is applied between the buffer well 116 and the buffer waste well 114. This leads to the movement of the amplified nucleic acids 23 captured in the transport channel 102 and the detection (CE) channel 112 along the pathway connecting these wells.

[0191] Application of a strong electric field 131 causes separation of the DNA molecules based on molecular size. In the detection channel 112 further downstream from the initial location of the DNA in the CE channel 112 (e.g., 75 mm from the point where the DNA 23 is trapped between the buffer well 116 and the buffer waste well 114), fluorescence can be stimulated by appropriately illuminating the separated species 140 with a laser source or LED or other light source with a wavelength that matches the excitation wavelength of the fluorophore attached to the species 140. One of skill in the art can readily ascertain the appropriate wavelength to use based on the fluorophore selected.

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

[0193] The separation module 29 of the chip 20 (FIG. 4) can be advantageously utilized as a stand-alone module for a number of other applications, including DNA, RNA, and protein analysis. To tailor the chip to a specific application, it may be necessary to manipulate a combination of flow parameters to obtain the desired level of analytical data (i.e., high resolution in the separation).

[0194] In any of the embodiments described herein, the length of the detection channel 112 can be adjusted from a few centimeters to several meters to accommodate the separation of a large number of individual molecules, which 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 thousands of volts to achieve the desired level of fraction (DNA / RNA / protein) separation. This improves the ability to resolve the separated molecules. One skilled in the art can ascertain the electric field to be applied based on the length of the detection channel 112 (FIG. 4), the desired resolution, and the molecules of interest.

[0196] Those skilled in the art will also understand that different gel media are 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, a low concentration of acrylamide can be used to separate high molecular weight proteins (e.g., 5% for 40-200 kDa), while a high concentration of acrylamide can be used to separate low molecular weight proteins (e.g., 15% for 10-40 kDa). Similarly, a low concentration of agarose can be used to separate high molecular weight nucleic acids (e.g., 0.5% for 1-30 kbp), and a high concentration of agarose can be used to separate low molecular weight nucleic acids (e.g., 1.5% for 0.1-2 kbp).

[0197] Additionally, non-optical detection strategies, such as electrochemical detection strategies, may be used, alleviating the need to fluorescently label molecules that are detected by separation.

[0198] Kits and preloaded integration chips In an exemplary embodiment, different integrated chips can be prepared for analyzing different pathogens, diseases and biological samples.

[0199] An end user of the integrated chip described herein can analyze various biological samples by configuring the chip and hardware system (described below) at each location as described above with reference to Figure 10. To further enable this capability, the integrated chip of the present invention can be included in a kit that further comprises components such as reagents including various of the above-mentioned buffers, molecular markers, molecular weight standards, magnetic beads, oligonucleotides, fluorescent dyes, CE sieving matrices, etc. Alternatively, many of these components can be preloaded onto the integrated chip.

[0200] Referring to FIG. 4, an integrated chip 20 can be fabricated with at least one extraction chamber 104, which includes magnetic beads that can be coated with, for example, streptavidin. In some embodiments, the at least one extraction chamber 104 can include reagents for cell lysis and nucleic acid extraction (e.g., lysis buffer as described above). In some embodiments, the at least one extraction chamber 104 can include microfabricated protrusions disposed within and / or within the at least one transport channel 102, which can serve to mechanically shear and lyse cells of the biological sample. At least one amplification chamber 108 can be preloaded with magnetic beads, oligonucleotides that may be fluorescently labeled, amplification master mix, etc. At least one detection channel 112 can be preloaded with CE sieving matrix and / or running buffer and / or molecular weight standards. In another embodiment, at least one detection channel 112 includes a hybridization microarray for identification of 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 at least one detection channel 112. In other embodiments, the integrated chip may include at least one fluid control valve that controls the flow of fluid through at least the microfluidic channel. The valve may be a passive plug or any of the valve systems described below.

[0202] Precision Control The embodiments according to the invention utilize nano-manipulation and precision control of molecules at the nanoscale. By controlling molecules or fluidic systems with ultra-high precision, the embodiments according to the invention can dramatically improve the overall quality of chemical and biochemical reactions carried out under highly controlled and tunable conditions on nano-engineered chips or precision-engineered chips. Thus, by utilizing nanotechnology, the embodiments according to the 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 the nano-engineered chips according to the embodiments of the invention and the platform devices according to the embodiments of the invention tighten the overall Gaussian spread in the products produced by the on-chip reactions, allowing for orders of magnitude improvement across several important performance metrics including speed, accuracy, deployability, adaptability and robustness.

[0203] The embodiments according to the present invention allow for precision or nanoscale control of nucleic acids and proteins. Nanoscale or precision reactors allow for unexpected improvements in the time, cost, speed and infrastructure required to perform analyses of biological samples compared to conventional larger scale systems. Tension can be applied to nucleic acids, for example by electromagnetic fields acting on the reaction chamber, which can cause nucleic acids, such as 2 nm wide DNA molecules, to stretch in a given direction. This increases the rate at which reactions tend to occur, for example by opening the DNA. In this way, small scale molecular control is achieved. Such small scale control allows for the achievement of greater signal to noise ratios, improved yields, and reduces the time to equilibration in the reaction chamber. A variety of forces can be used to control at the nanoscale, for example mechanical, electromagnetic, and / or hydrodynamic forces, and the use of the types of tension forces taught in U.S. Pat. No. 7,494,791, the entire disclosure of which is incorporated herein by reference.

[0204] The phrase "precise control" of a parameter as used herein refers to the ability to control the parameter so that repeated measurements of the parameter under unaltered conditions show the same results within a precision range, and further refers to the ability to adjust the parameter to a new value of the parameter within such precision range. For example, according to an embodiment of the present invention, a parameter governing at least one step of a reaction used to analyze a biological sample can be precisely controlled to 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 can be adjusted to a new value of the parameter with such a precision range. Such precise control can be performed in a number of ways, including, for example, using controlled fluid flow or applying tension, and includes precise control for both proteins and nucleic acids.

[0205] As used herein, applying tension to a nucleic acid or protein includes the direct or indirect application of a force to the nucleic acid or protein that tends to stretch or elongate the nucleic acid or protein. By way of example, tension can be applied to the nucleic acid or protein by direct application of mechanical tension, hydrodynamic stress in a fluid flow, or electromagnetic fields, whether acting on the nucleic acid or protein molecule itself and / or on a surface, substrate, particle, etc. to which the nucleic acid or protein is bound.

[0206] According to an embodiment of the invention, tension tending to stretch the nucleic acid may be applied in at least one cycle. Further, tension tending to stretch the nucleic acid may be applied and may be varied during each cycle of the following steps (a)-(e) in a nucleic acid amplification method: (a) contacting one or more template strands of single-stranded nucleic acid with one or more oligonucleotide primers that are complementary to a portion of the one or more template strands; (b) annealing at least one of the one or more primers to said portion of the one or more template strands that is complementary to the primer; (c) contacting one or more template strands with a nucleic acid polymerase and at least four different nucleotide triphosphates; (d) extending at least one annealed primer with a nucleic acid polymerase, thereby forming one or more extension products bound to one or more template strands; (e) separating one or more extension products from one or more template strands; and (f) repeating steps (a), (b), (c), (d) and (e) to amplify the nucleic acid, wherein at least one of the one or more extension products of step (e) is used as a template strand in a next cycle of steps (a) to (e); Here, in the final cycle, step (e) may or may not be included.

[0207] Additionally, at least one cycle of step (b) or (d) includes applying tension to one or more template strands that tends to elongate the nucleic acid. In some versions, the treatment can be carried out at a constant temperature.

[0208] According to embodiments of the present invention, precise control can 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 forces, such as those 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 can be used.

[0209] The level of precision control can be on the nanometer scale, for example, where DNA can be 2 nm in diameter and proteins 10 nm in diameter. According to embodiments of the invention, control of the translational position or rotational orientation of molecules can be achieved to within less than 100 nm, less than 10 nm, or less than 1 nm. Furthermore, the reaction chamber itself can be small, such as 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 its largest dimension. Furthermore, temperature, pressure, diffusivity, tension, chemical concentration, salt concentration, enzyme concentration, or other parameters that regulate or govern the reaction can be varied within tightly controlled ranges, 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] As used herein, when a system is "configured" to perform precise control of a parameter, the control of such a parameter may be performed, for example, using one or more software or hardware modules executed under the control of a computer processor of a mobile device according to an embodiment of the present invention. The computer processor may include one or more control modules specifically programmed to receive digitally coded signals corresponding to the values ​​of one or more controlled parameters and to perform closed-loop control of these controlled parameters. For example, the control module may receive the values ​​of the parameters and adjust one or more forces or other conditions applied to the molecules being analyzed based on the values ​​of the parameters (e.g., using applied tension). The adjustments made by the control module may result in a change in the parameter, thus forming a closed feedback loop under which the control module performs closed-loop control of one or more controlled parameters. In addition to receiving digitally coded signals from one or more other computer modules, the control module may receive one or more sensor inputs from one or more sensors that sense the values ​​of the controlled parameters; for example, a temperature or pressure sensor may provide a temperature or pressure sensor input to the control module.

[0211] Hardware System As mentioned above, the compact integrated chip with biological assay is intended to be disposable. The present invention also includes hardware systems that include 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 accommodate chips of various designs that utilize the above biological processing modules (e.g., nucleic acid extraction, thermal or isothermal amplification, laser-based fluorescence detection and CE separation).

[0212] 11 is a block diagram of a hardware system 400 included in the present invention. Details of the configuration of the hardware system 400 are further described below with reference to FIG.

[0213] 11, the chip 20 (which may be disposable) is typically inserted into a hardware module 400 that is controlled by a microprocessor / computer interface 408. As shown in FIG. 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 accommodate the processing that follows sequentially on the chip 20, or each of the modules 402, 404, and 406 may be controlled to perform the operations of any one or a combination of two modules. The respective characteristics of each hardware module 400 are as follows:

[0214] The microprocessor / computer interface 408 means may comprise, 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 storing operating and signal processing software suitable for detecting nucleic acids, such as any of those taught herein.

[0215] Extraction Control Module Referring again to FIG. 11, a 12,000-13,000 Gauss magnet or electromagnet is added below or above the extraction chamber (chamber 104 shown in FIG. 4) in the extraction module 402, if desired. The application of the magnetic field can be controlled for anywhere from just a second to tens of minutes, allowing for aggregation and retention of the magnetic beads as desired by the protocol. If desired, heat can be used to elute the DNA from the beads, as described in the extraction protocol. Thus, in addition to the application of a magnetic field, application of heat of about 50-90° C. for any suitable length of time during amplification can be accomplished by the hardware system 400 using the same thermal module used in the amplification module 704 described next.

[0216] Amplification Control Module In FIG. 11, the temperature of the amplification module 404 is cycled between about 40-95° C. for thermal cycling. This is accomplished using a Pelletier device or any other suitable heating / cooling device that may be used for amplification. Up to three thermal cycle temperatures allow for DNA denaturation (70-100° C.), primer annealing (about 40-80° C.) and extension temperatures (50-90° C.) for desired time intervals and repeated cycles to create exponential DNA amplification. The designated amplification chambers on the chip 20 are placed over a Pelletier device that allows for temperature cycling.

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

[0218] Detection Control Module 13 is a block diagram of the detection control module 406 in accordance with the system of the present invention, which utilizes laser-induced fluorescence in combination with high-voltage electrophoresis for detection of amplified DNA or, in some embodiments, separated proteins. The detection control module 406 uses a programmable high-voltage source 620 to provide the necessary voltage signals (400V-10 kV) via electrodes 622 along designated channels on the chip 20. A safety feature is built in that shuts down the high-voltage source 620 if the enclosure (not shown) housing the electronics and chip does not close and stabilize.

[0219] A laser source 602 with the desired wavelength and appropriate filter arrangement (not shown) illuminates a selected portion of the chip 20 through a lens 606. The illumination excites fluorophores bound to the DNA or proteins, causing them to emit radiation (not shown) at a wavelength different from the laser wave. The filter blocks the laser light and transmits the emitted radiation, which is collected by a lens 607. The lens 607 focuses the filtered radiation onto a detector 608, such as a photomultiplier tube, avalanche light emitting diode, charged coupled device, or other suitable detector. The detector emits a signal, such as a photocurrent, which is amplified (and optionally filtered) by a signal amplifier circuit 610. A computer 408 records and processes the amplified signal; and the computer 408 may be used to control the high voltage source 620 and the laser 602 with a software interface, as follows. Those skilled in the art will appreciate that alternative arrangements of optical elements may be used to interrogate fluorescent markers bound to DNA.

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

[0221] Cover 754 may be hinged or positioned over the chip, which is held in groove 758 of holder 752. By placing cover 754 over the chip, electrodes 776 are inserted into the appropriate wells of the chip (e.g., CE wells 114, 116, 118, and 120 of chip 20 of FIG. 4). Electrodes 776, coupled to high voltage source 620 controlled by computer 408, may apply electric fields of different strengths (e.g., 400-6000V) across corresponding portions of the chip, such as CE channel 112 shown in FIGS. 4 and 8A-8C.

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

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

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

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

[0226] Additionally, the GUI provides the user with the ability to (1) select and power any pair of electrodes configured for application of a high voltage signal; (2) cycle through the above selections for a given time interval as desired by the user; (3) obtain feedback monitoring the voltage applied across any of the powered electrode pairs and graph this data as a function of time; (4) obtain feedback monitoring the current through any of the powered electrode pairs and graph this data as a function of time; (5) graph the acquired fluorescent signal as a function of time; and (6) identify / distinguish the fluorescent signal (peak / intensity) from the size standard signal. These features may be used to confirm amplification and identify the presence (or absence) of sequences in the DNA.

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

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

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

[0230] In other embodiments, various valve systems may be used, such systems being described elsewhere herein, see, for example, Figures 5, 6A-6B, and 7A-7B.

[0231] Yet another embodiment of an integrated microfluidic chip includes a valve technology called "elastic valves," 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 catheters used in medical surgery. The fluid path includes a channel 1102 formed between an upper chip substrate 1104 and a lower substrate 1106. Inflation or actuation of the membrane 1110 can be achieved with an inlet air tube 1112 connected to the membrane 1110 (i.e., the membrane 1110 is pneumatically actuated and controlled) as shown in Figures 15A and 15B. When the deflated membrane 1110 is placed in an open port 1108 as shown in Figure 15A, fluid flow 1114 is possible across the channel 1102. However, as shown in Figure 15B, when the elastic valve is inflated with air 1116, it expands to seal the open port 1108, thus restricting the flow of fluid 1114 across the open port 1108 of the channel 1102. In an integrated chip, a series of such valves 1100 may be used to achieve flow control as needed.

[0232] In another operating configuration, the elastic valves can be embedded within the chip, rather than being inserted or operated at an external interface, to achieve a higher level of automation. An example of such a configuration would include a fabrication process in which an elastic membrane is sandwiched between two layers of the chip along any channel requiring fluid control. The inlet air tubes used to actuate the elastic membrane valves are also embedded within the chip, and brought to the periphery of the chip for connection to external pneumatic lines. Both embedded and external valve and pump systems can be effectively used to regulate volumes ranging from nanoliters to several milliliters using the valves / pumps.

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

[0234] As shown in Figures 16A-16F, fluid can be drawn from the open well 1202a to the closed chamber 1208 by sequentially expanding and contracting the three membranes 1204a-1204c. First, fluid is loaded into the open well 1202a as shown in Figure 16A. The elastic membranes 1204a-1204c are then sequentially expanded as shown in Figures 16B-16D. Once all three membranes 1204a-1204c are expanded, the membrane 1204a in the channel 1206a is contracted, creating a suction force that draws fluid from the open well 1202a into 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. Fluid can also be unloaded from the closed chamber 1208 into either of the open wells 1202a and 1202b by sequential operation of similar membranes 1204a-c.

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

[0236] In one embodiment, the system may be used in a method for implementing personalized medicine. In a broad sense, personalized medicine uses genetics to provide the ideal patient with the ideal drug at the ideal dose for the ideal outcome. In an embodiment according to the present invention, a portable assay system is used to extract, amplify and detect nucleic acids from a sample, in particular to detect personalized biomarkers based on nucleic acids. The system may then determine the appropriate dosage and / or drug combination for customized drug delivery based on the detected personalized biomarkers. Targeted drugs and companion diagnostics may be provided. Furthermore, the system may be used to determine whether a person will respond to drug treatment. The system may also be used to facilitate stratifying patients, improving drug safety and efficiency, and optimizing dosing and treatment regimens. Furthermore, the system may be used to monitor a person, for example, by monitoring the amount of nucleic acid found in a person's biosamples taken at different times. Such monitoring may 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 may be diagnosed, classified or monitored via DNA / RNA markers, such as inflammatory markers. For example, determining an individualized genomic profile can include detecting a nucleic acid indicative of a type or subtype of diabetes.

[0237] In another embodiment, the portable system according to the embodiment of the present invention can be used to enrich experimental groups, helping to reduce the size and cost of regulatory clinical trials. Individualizing tests with subset gene groups can dramatically improve therapeutic efficacy and shorten the approval process. The resulting drug and companion diagnostic combinations have additional value for the target gene groups.

[0238] In another embodiment, the portable system according to the present invention can be used to provide personalized care, for example in the field of cosmetics, cosmeceuticals, and skin care applications. For example, the portable assay system can be used to extract, amplify, and detect nucleic acids from a sample; in particular, to detect personalized biomarkers based on the nucleic acids. The system can then determine the type, amount, or combination of personalized care products to deliver based on the personalized biomarkers. For example, the system can be used to select and deliver cosmetics based on personalized beauty biomarkers. In one example, the skin type can be determined based on personalized biomarkers, which can then be used to determine the type, amount, or combination of beauty products to deliver to a person. The mobile device can be used to measure and quantify the presence of key biomarkers (e.g., which can be DNA or RNA based) in real time. Sequence biomarkers (e.g., beauty biomarkers such as age-related loci, specific aging genes or gene expression patterns, skin quality) can be measured for skin products. The portable system according to an embodiment of the present invention can be used to correlate an individual's genotype with such sequence biomarkers. The integrated chip can be customized to follow a library of target genes for beauty biometrics. An individual's beauty biometrics can be measured by quantifying the individual's beauty biometrics in real time via a portable system according to an embodiment of the present invention. It can then be seen how these biomarkers change over time and with the use of corresponding beauty products. The embodiments can also be used for wellness applications, for nutrigenomics, and for Ayurvedic genomics to perform Ayurvedic diagnosis via genes corresponding to, for example, Vata, Kapha, and Pitta body types.

[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 combined with a specific and uniquely determined pharmaceutical product. For example, the portable assay system may be used to extract, amplify and detect nucleic acid of a sample; in particular, to detect a personalized biomarker based on the nucleic acid, where the personalized biomarker may indicate the presence of a specific strain of a disease or pathogen. The system may then uniquely 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 for a specific strain of human immunodeficiency virus (HIV). The portable system according to the present invention may be used for point-of-care detection of HIV strains, HIV viral load determination and HIV genotyping, or as a drug monitoring device or tool to monitor and prevent mother-to-child transmission of HIV.

[0240] More generally, embodiments according to the invention may be used to identify the genotype of any organism, thereby determining at least one of: predisposition to genetic disease, strain of disease, and antibiotic resistance of a disease state, for example, to determine the type of viral hepatitis; or to identify cancer genes.

[0241] In various embodiments, the portable system according to the present invention may be used in a variety of different possible industries. For example, the portable assay system may be used to extract, amplify and detect nucleic acids from a sample. The detected nucleic acids may then be used in any of a variety of different possible industries (in addition to the industries discussed elsewhere herein). For example, the detected nucleic acids may be related to food safety, agricultural diseases, veterinary applications, archaeology, forensics, nutrition, dietary supplements, nutrigenomics, water testing / satellite, food and beverage, environmental monitoring, customs, security, defense, biofuels, sports and wellness; and may be used for theragnosis.

[0242] The system according to the embodiment of the present invention can be used to determine the personalized genomic profile of the person from whom the biological sample is derived. The personalized genomic profile can then be used to personalize the person's dietary supplements, cosmeceuticals, pharmaceuticals, food or beverages, or nutritional regimen based on the personalized genomic profile. The personalized genomic profile can be used, for example, for personalized wellness, personalized sports nutrition, personalized diet, or personalized nutrition for the person. In the field of sports, for example, inflammatory markers can be determined to detect, measure, and treat injuries in athletes, such as traumatic brain injuries in athletes in contact sports. In another embodiment, markers can be determined to evaluate blood doping in sports. In another embodiment, various types of phenotypic expression of RNA markers can be measured.

[0243] The system according to the embodiment of the present invention can be used for personalized nutrition, where the chip reader (such as the nucleic acid extraction control module, the nucleic acid amplification control module, and the 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 involves using genetics to provide the ideal nutrition to the ideal consumer in real time for ideal results. In the embodiment according to the present invention, a companion personalized nutritional additive can be provided to a person based on the DNA / RNA profile. Targeted nutrition and companion diagnostics can be provided, and companion diagnostics for health and wellness biomarkers can be developed. A holistic and integrated approach to health and wellness can be provided. Feedback can be provided to the consumer to measure progress not only by commonly accepted phenotypic markers, but also by DNA / RNA biomarkers that provide a robust and reliable measurement of progress. Determining a person's genetic profile or predisposition based on DNA / RNA markers can personalize the person's diet. The personalized genomic profile can be used to determine a person's nutritional predisposition, such as genetic food allergies, intolerances or sensitivities, e.g. gluten sensitivity, gluten intolerance or lactose intolerance, and diets can be personalized based on the profile. Inflammatory markers can be measured as a way to monitor tolerance or non-tolerance of a particular diet. Furthermore, nutritional disorders can be detected, measured and then addressed by adding micronutrients to foods or beverages. For example, vitamin, mineral and other deficiencies (such as vitamin B12 deficiency, folic acid deficiency, iron deficiency) can be measured based on the biomarkers measured by the embodiments according to the present invention. The deficiencies can then be addressed by providing the appropriate micronutritional supplement to the food or beverage. Furthermore, probiotics or prebiotics that are deficient or altered in the person's microbiome can be determined.For example, a biological sample from a person's saliva, gastric juices, or urine can be obtained and analyzed to determine what probiotics or prebiotics are deficient or altered in the person's microbiome, such as the person's oral microbiome or stomach microbiome. The deficient probiotics or prebiotics can then be replenished in the person's food or beverage to help restore the person's normal microbiome.

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

[0245] In another embodiment according to the present invention, a consumer experience of a food or beverage may be enhanced by dispensing the food or beverage based on a person's nutritional genomic profile. The system taught herein may be embedded in a dispensing activator form factor, such as at least a portion of a vending machine, automated teller machine, or kiosk, that may be controlled based on a control input from the system taught herein. For example, a person may provide a biological sample to a genetic analyzer configured to determine biomarkers associated with the person. Based on the biomarkers, a personalized genomic profile of the person from whom the at least one biological sample is sourced may be determined. The dispensing controller is then configured to determine a product or a portion of a product to dispense based on the personalized genomic profile. The dispensing activator is coupled to the dispensing controller and configured to dispense at least a portion of the product under the control of the dispensing controller. With such a system, for example, a consumer may insert a biological sample, such as a saliva sample, into a vending machine that may 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 three-dimensional printer configured to print at least a portion of the product, or the dispensing activator may be configured to dispense a prepackaged product. For example, the product may be a food, beverage, cosmeceutical, nutraceutical, pharmaceutical, herbal, alternative medicine, and cosmetic. The dispensing controller may be configured to determine the product or portion of the product to dispense based on nutritional predisposition, nutritional disorder, and at least one probiotic or prebiotic deficient or altered in the microbiome, any of which may be determined by detecting nucleic acids according to embodiments taught herein. For example, a three-dimensional printing machine may be used to print the consumer's food or beverage at a vending machine or kiosk based on the consumer's genetic profile. A user interface, such as a keyboard, touchpad, speech recognition interface, augmented reality interface, or other user interface, may be used by the consumer to input additional information.A consumer may be given some information about alternative ingredients that have benefits based on the consumer's profile, for example, and then be able to select one or more of the ingredients using a user interface to make the food or beverage to order. Systems such as vending machines may integrate point-of-use companion diagnostics and point-of-use production of fortified foods or beverages, for example, using 3D printing of the food or beverage. Foods, beverages, cosmeceuticals, dietary supplements, and cosmetics may be printed in 3D in this manner. In cosmetics, for example, a customer may be provided with a customized profile at a cosmetics counter in a kiosk using the system taught herein, and then a custom cosmetic product, such as a skin care solution, may be printed. Similar systems may be provided for dietary 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 provided in juice to reduce the consumer's inflammation levels. Customized dosages may be provided by a control system based on the customer's genetic profile in the next embodiment.

[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 are monitored in real time by the nucleic acid analysis system taught herein and used to provide electronic control input to the EECP machine, for example, to increase or decrease blood flow or titrate medication. For example, inflammatory factors, VEGF, endothelial growth factors, other gene expression patterns, cardiac markers, DNA / RNA markers that correlate with cardiac status, etc. may be monitored by the system taught herein. Coupling the nucleic acid analysis system taught herein with an EECP therapy machine may provide a tool to help reverse atherosclerosis with the combined effect of stimulating endothelial repair with the measurement of flow and inflammatory factors. Biomarkers related to the atherosclerotic process may be monitored simultaneously on multiple channels of the nucleic acid analysis system, and electronic control input may be provided to the EECP machine based on the amount of biomarkers.

[0247] In various embodiments, the results provided by the portable systems according to the invention include viral load (e.g., copy number per ml), predicted cell count per volume (e.g., mm 3 The results may include predicted CD4 counts in cells per 100 μg / mL and titration of drug dosing.

[0248] In further embodiments according to the invention, the portable system according to the invention may communicate with other systems in a variety of different possible ways. The portable system may send and receive regulated data signals related to the biological sample, and may communicate via wired media (e.g., wired network or direct wired connection) or wireless media (e.g., acoustic, infrared, radioactive, microwave, spread spectrum). The portable system may communicate, for example, via 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 that stores genome profiles. The portable system may store or send or receive signal profiles of a single reference sample.

[0249] In further embodiments according to the invention, the portable system or mobile device may utilize a compact integrated chip, even disposable, to be implemented as part of or interface with any of a variety of different possible handheld or tablet devices widely available, such as a smart phone, personal digital assistant (PDA), mobile phone, or other handheld or tablet device. Furthermore, a similar graphical user interface and external design may be used as used on such widely available handheld or tablet devices. In one example, an embodiment according to the invention may be implemented with, interface with, or use a graphical user interface or external interface similar to that of the iPhone, iPad, or iPod sold by Apple Inc. of Cupertino, California, USA, or the Galaxy sold by Samsung Electronics Co., Ltd. of Suwon, South Korea, all of which are incorporated herein by reference.

[0250] In an embodiment according to the invention, a portable system or multiple such portable systems at distributed locations may be used to track disease outbreaks at widely distributed assay sites. 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 two-way information exchange between a central data center and the system end users. The data center may provide known pathogen / disease mapping information to the end users / invention systems for biological sample analysis, and the inventive systems / end users may then transmit assay results to the data center. The data center may receive geographic location information and other case specific information from the end users / invention systems. The data center may monitor assay results coming from multiple deployed departments / invention systems and utilize pattern detection programs to track, for example, disease outbreaks. The data center may programmatically generate notifications to remote portable systems according to the invention upon detection of threshold patterns.

[0251] Further Uses and Applications of the System and Integrated Chip In various embodiments, the systems of the invention may be used to identify pathogens, diagnose diseases with genetic markers, or genotype individuals. The methods of the invention generally include: (1) providing at least one integrated chip; (2) loading at least one biological sample onto the at least one integrated chip; (3) operably linking a portable control assembly with the at least one integrated chip; and (4) activating the portable control assembly to perform nucleic acid extraction, amplification, and detection of the biological sample loaded onto the integrated chip.

[0252] The present invention may be used to diagnose and detect a wide variety of pathogens and disorders that have nucleic acid-based genetic material and / or genetic components. In addition to detecting targets such as HIV, HBV, HCV and sexually transmitted diseases, the systems and methods of the present invention may be used to detect and diagnose molecular diagnostic targets arising in the fields of oncology, cardiology, specific testing and prenatal screening.

[0253] Preferably, the biological sample is derived from a biological fluid such as, but not limited to, blood, saliva, semen, urine, amniotic fluid, cerebrospinal fluid, synovial fluid, vitreous fluid, gastric fluid, nasopharyngeal aspirate and / or lymphatic fluid.

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

[0255] Pathogens detectable by the systems and methods of the present invention can be from a variety of hosts. The host, whether living or non-living, must be capable of supporting the replication of the infectious agent by allowing the infectious agent to replicate in or on the host. Examples of such hosts include liquid or solid in vitro culture media, animal cells or tissues, plants or single-cell organisms, all organisms including mammals such as humans.

[0256] The system and method of the present invention may be utilized in one of many of the following areas: In one embodiment, the system and method of the present invention may be utilized in the area of ​​defense against biological weapons. For example, the present invention may be used for pathogen detection at the point of emergence and in real time. In another embodiment, the system and method of the present invention may be utilized in the area of ​​life sciences. For example, the present invention may be used as and in conjunction with portable analytical instruments. In another embodiment, the system and method of the present invention may be utilized in the area of ​​clinical diagnostics. For example, the present invention may be used by doctors, nurses or untrained users in hospitals, at home or in the field to diagnose and / or identify pathogens. The present invention may also be used to identify the genotype of an organism, thereby determining predisposition, if any, to genetic disease or predisposition, if any, to antibiotic resistance. The present invention may also be used to determine the pathogens present in a patient and their susceptibility and resistance profile to various antibiotics. The present invention may also be used as a drug monitoring device, a prognostic indicator of disease, and a diagnostic treatment device. In another embodiment, the system and method of the present invention may be utilized in the area of ​​industrial and agricultural monitoring. For example, the present invention may be used to monitor and / or detect pathogens borne in food, crops, livestock, etc. In another embodiment, the systems and methods of the present invention may be used in the area of ​​forensics. For example, the present invention may be used to genetically identify individuals.

[0257] In one embodiment, genetic disorders and disorders with genetic components can be diagnosed by using the system and method 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 breast cancer progression and metastasis; and BRCA1, which is involved in 50% of all hereditary breast cancers and is also involved in the increased risk of prostate cancer and other cancers.Screening for these genetic markers can be achieved using the system and method described herein.

[0258] Infectious pathogens that may be diagnosed using the systems and methods 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 undulant fever; Yersinia; Pasteurella; Francisella; Actinocacillosis; Haemophilus, Bordetella (e.g., Burdetella pertussis;Pseudomonas and Legionella;Bacteroides, Fusobacterium, Streptobacillus and Calymmatobacterium;Bacillus (spore-forming aerobes);Clostridium (spore-forming anaerobes);Lissteria and Erysipelothrix;Corynebacterium;Mycobacterium;Spirochetes;Rickettsias;Chlamydia;Mycoplasma;Poxviruses;Herpesviruses;Papovaviruses;Adenoviruses;Orthomyxoviruses, Paramyxoviruses;Rhabdoviruses;Cytomegaloviruses;Retroviruses;Picornaviruses;Cornaviruses;Rotaviruses;Hepatitis viruses (e.g. Hepatitis C virus, Hepatitis B virus);Togaviruses;Buniyaviruses;Arenaviruses;Cryptococcus;Candida (e.g. Candida albicans, Candida glabrata, Candida Parapsilosis, Candida tropicalis); Sporothrux; Ilestoplasma; Coccidioides; Blastomyces; Aspergilli; Zygomycetes; Dematiaceae; Fusarium; Protozoa; Nemathelminthes;

[0259] In other embodiments, retroviruses such as HIV-1, HIV-2, any of HIV-1 groups M, N, O, or P, any of HIV-1 group M subtypes A-K, or other known types or subtypes of any of HIV, HTLV-1, and HTLV-2, and herpes viruses such as HSV-1, HSV-2, VZV, EBV, CMV, and HHV-6 may be detected using the systems and methods of the present invention.

[0260] In other embodiments, disorders and diseases that may 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 (genera, 6, 11, 16, 18, 31, 33), Moraxella catarrhalis, Streptococcus pneumonia, Haemophilus influenza, Legionella spp.; human parvovirus B19, Group B streptococcus.

[0261] In other embodiments, disorders and diseases that can be detected using the systems and methods of the present invention include genetic diseases and disorders with a genetic predisposition, including, but not limited to, cystic fibrosis, Gaucher disease, medium-chain acyl-CoA dehydrogenase deficiency, myotonic dystrophy, sodium channelopathies, chloride channelopathies, 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 agent detected is methicillin-resistant S. aureus (MRSA). Staphylococcus aureus is one of the most prominent pathogens causing nosocomial and community-acquired infections. Beta-lactam antibiotics are the preferred agents for serious S. aureus infections. Since the introduction of methicillin in clinical practice in 1961, there has been a steady increase in methicillin-resistant S. aureus (MRSA) strains, making nosocomial infections a serious worldwide problem. Thus, detection of methicillin resistance has important implications for patient treatment and management. In clinical laboratories, S. aureus is identified by growth characteristics followed by detection of catalase and coagulase activity or specific surface components. DNase and thermostable endonuclease tests are used as confirmatory tests for indeterminate or negative coagulase tests. Conventional susceptibility testing of S. aureus reliably detects resistance to methicillin or oxacillin when agar dilution or agar screening methods are used according to NCCLS standards. Methicillin resistance involves the production of penicillin-binding proteins encoded by the mecA gene or, in rare cases, is associated with overproduction of beta-lactamases.

[0264] Traditional 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 and problematic, with false positives and false negatives being common. These deficiencies are due to the expression of staphylococci resistance genes, which can be expressed in a very heterogeneous manner, making characterization of the resistance genotype difficult. Molecular methods can be used that target the detection of the resistance gene mec A. Screening for mutations in the amplification products can be aided by the use of high-density probe arrays, such as Affimetrix® Genechips™, which can be utilized in the practice of the present invention.

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

[0266] In other embodiments, viral and microbial diseases that may be detected using the systems and methods of the present invention are those listed in Table 2: [Table 2]

[0267] In further embodiments, the infectious diseases listed in Table 3 may be tested for using the systems and methods of the present invention: [Table 3]

[0268] Multi-sector, industry-wide use Given the above features and advantages of the present invention, many developments are possible within the healthcare (and other industries), particularly across multiple sectors, including:

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

[0270] The commercial sector (e.g., hospitals / healthcare organizations, analytical / diagnostic laboratories, pharmaceutical entities, etc.), and

[0271] Private sector (e.g. doctors, farmers / ranchers, individuals).

[0272] In an exemplary embodiment, a laboratory or lumber company serves as a supply point for the various integrated chips prepared above. Different chips are prepared to analyze different pathogens and diseases. End users of the chips come from various sectors and deploy the portable system at each location and environment, including patient use at home, field (e.g. crop and veterinary checkpoints), remote / regional locations, as well as hospitals / medical centers, ports of entry, etc. Due to the plug-and-play configuration between the integrated chips and portable systems of the present invention (i.e., fast reader response time, chip assay versatility, and chip disposability), a given portable assay system / device of the present invention may read a variety of different chips (to detect different pathogens / diseases) at a location, or may be dedicated to reading one type of chip (to detect a specific pathogen / disease).

[0273] During use of the portable assay system, a two-way information exchange occurs between the central data center (where the operational data library and bioinformatics data reside or are maintained) and the system end user. In this two-way exchange, the data center may provide known pathogen / disease mapping information to the end user / invention system for biological sample analysis, and the invention system / end user may then transmit the assay results to the data center. In addition to the assay results, the data center may receive geographic location information and other case specific information from the end user / invention system. The data center may monitor the assay results coming from multiple deployed units / invention systems, utilizing pattern detection programs (common in the art). For example, based on data received from multiple end users, patterns of 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 of the occurrence of a particular condition or pathogen / disease (e.g., in water supplies, food crops, blood banks, etc.) are contemplated.

[0274] The data center, in turn, can be configured to programmatically generate notifications upon detection of the threshold pattern. The data center can notify medical personnel (ambulance, emergency room, physicians), military units, government agencies, etc. Such notifications can be on a subscription basis where the recipient pays a subscription fee to join the data center alerts and notifications. There can be different subscription levels where the party / user pays at one level to have access to the data library and pays at another level to access the collected assay result data. Another subscription level can provide automated alerts and notifications to the subscriber using contact information provided by the subscriber (where and in what medium / form to send the alerts).

[0275] As used herein, notifications and alerts can be in the form of emails, phone calls, alarms, or other audible and / or visual indications.

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

[0277] The present invention thus provides enhanced methods of medical care and treatment, disease control, biodefense (point-of-emergence and real-time pathogen detection, and real-time tracking of infection and contact transmission), and other uses across multiple sectors and industries.

[0278] Example Device Configuration The inventions described herein may be configured or used in products or devices including, but not limited to, handheld devices, computer tablets, notebook computers, smartphones, implantable devices, ingestible devices, wearable devices, and injectable devices.

[0279] A "wearable device" is a device intended for use on an external part of the body, such as an arm, a leg, skin, a cheek patch, a torso, etc. A wearable device is configured to be held on the body, such as, but not limited to, a chip-on, a bracelet, an adhesive patch, a skin patch, a cheek patch, glasses, a headband, an ankle band, a shoe accessory (on the sole of the foot), etc. An implantable device may routinely perform the operation of the device described herein and interface the output of the operation of the device with a computer, such as by wireless communication. A wearable device according to an embodiment of the invention may comprise one or more separate elements, which may or may not be attached or mechanically interfaced together and / or may be present on one or more different external parts of the body and may communicate with each other using wireless and / or wired communication. A wearable device according to an embodiment of the invention may also comprise only a portion of such a device herein and may communicate wirelessly and / or wired with other portions of such a device herein, which are internal and / or external to the human body. Exemplary wearable devices are shown in FIG. 21 and are configured as (A) a skin or dermal patch; (B) a bracelet comprising a device according to the present disclosure and a band for holding the device on the neck, arm, leg, or finger; and (C) a patch with one side adhesively attachable to the exterior of the body and the same or opposite side adhesively attachable to the device of the present disclosure. In some embodiments, all of the elements of the device are included on the wearable device. In other embodiments, some of the elements are included on the wearable device and are transmitted to a receiver located elsewhere. Wearable devices according to embodiments of the present disclosure may also comprise only a portion of the devices described herein and be in wireless and / or wired communication with other portions of such devices herein, which are inside and / or outside the human body.

[0280] An "implantable device" is a device that is placed into a surgically or naturally formed cavity in the human body where it is intended to remain in place for days, months, or longer. The implantable device may routinely perform the device operations described herein and may interface the output of the device operations with a computer, such as by wireless communication. An implantable device according to embodiments of the invention may comprise one or more separate elements, which may or may not be attached or mechanically interfaced together, and / or may be in one or more different parts of the human body and may communicate with each other using wireless and / or wired communication. An implantable device according to embodiments of the invention may also comprise only a portion of such a device herein and may communicate wirelessly and / or wired with other portions of such a device herein, which are inside and / or outside the human body.

[0281] An "ingestible device" is a device that can be orally delivered to a patient for personal monitoring. For example, the device can be an ingestible event marker used to time and record events logged by the patient using the devices described herein. The ingestible element wirelessly links through communication to an external recorder that records the date and time of ingestion and the unique serial number of the ingestible device.

[0282] An "injectable device" is a device that can be injected into a site within the human body and is capable of retention at an intended site within the body. The injectable device can routinely perform the device operations described herein and may interface the output of the device operations with a computer, such as by wireless information transfer.

[0283] An exemplary device, whether implantable, injectable or ingestible, is shown in Figure 22. The device of the present invention is incorporated into a carrier substrate.

[0284] Example DNA purification procedure

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

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

[0287] The user seals the amplification chamber 108, preferably using a passive plug valve that inserts into the reagent addition port 106 and sample well 120. Once the chamber is sealed, the extracted DNA mixes with the PCR master mix before a Pelletier cooler 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 propels the amplified nucleic acid to the detection module using a manual pump syringe connected to the integrated chip via the reagent addition port 106.

[0288] The user loads the polymer gel / sieving matrix and CE running buffer into the eight detection channels 112 via the buffer wells 116. Next, the molecular weight standards are loaded into the sample wells 120. The user then applies 400V across the electrodes located on or near the sample wells 120 and the sample waste wells 118, drawing the amplified nucleic acid into the detection channels 112. Once a sufficient amount of amplified nucleic acid has entered the detection channels, the user turns off the 400V field and then applies a 6kV field from the buffer waste well to the buffer well. This causes the molecular weight standards to separate along the detection channels 112, providing a time reference. The detection channels 112 may be allowed to flow, after which the user repeats the process with the loaded amplified nucleic acid forced into the sample wells 120 by syringe pressure applied to the nucleic acid amplification chamber 108. This causes the amplified nucleic acid to separate into species that travel downstream down the detection channels 112.

[0289] As they move, the molecular weight standards or amplified nucleic acid species pass through a measurement area 1005 where they are interrogated under the control of a detection control module 60 configured to detect a fluorescent signal, as shown in FIG. 9B. As the species 140 move through the measurement area 1005, they are illuminated by a laser beam 1004 of an appropriate wavelength, which causes the species 140 to emit a fluorescent beam 1010 of a different wavelength. The fluorescent beam 1010 and the laser beam 1004 exit the measurement area 1005 and propagate to a dichroic beam splitter 1007, which reflects the laser beam 1004 and transmits the fluorescent beam 1010. A beam block 1008 absorbs the laser beam 1004, and a detector 1012 senses the fluorescent beam 1010. The detector 1012 emits a photocurrent (not shown) commensurate with the intensity of the fluorescent beam 1010. A processor 1014 detects, records and processes the detected photocurrent as a function of time.

[0290] The detected and processed signal is matched against signals from known references analyzed under identical conditions to identify the pathogen in the sample. The system represents this identification process in an output signal graph 1020. Analysis of the known references provides a reference signal trace 1021 with peaks corresponding to known amounts of known pathogens. Comparing the time axis of the unknown sample's signal peaks 1022 to the reference signal trace 1021 provides the user with an indication of the type of pathogen; the system determines the viral load by calculating the area under the signal peaks 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 grown overnight in 5 mL test tubes in LB medium (Invitrogen) at approximately 37°C on a shaker at 200 rpm. The stock culture was then either used directly for the experiments or diluted (in RNase-free water) to obtain the desired cell dilution / concentration. The cultivated cell concentration was obtained by measuring the culture in a spectrophotometer (Ultraspec 10, Amersham Biosciences, UK). The details of E. coli species were as follows: E. coli, DH10B cells (ElectroMAX™ DH10B™, Cat. No.: 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 total genomic DNA was about 4.6 × 10 6 bp.

[0293] Using these cells, experiments were performed on-chip. Two sets of experiments are shown below. In the first set, stock cultures (0.2-2 μL) were used directly for the chip experiments. In the second set of experiments, 1 μL of stock culture was serially diluted up to infinite dilution. In each of these experiments, measurements were made during the course of the biological assay for both cell and DNA concentration, thus allowing quantified data for analysis. Cells were quantified as described above, while DNA concentration was performed on a Nanovue® (GE Health Sciences) spectrophotometer. However, due to the detection limits of both spectrophotometers used, quantification of low concentration samples was difficult until completion of the biological assay, which involved amplification allowing for easily quantifiable DNA amplification. Using the protocol described above, the following experiments were performed on the integrated chip.

[0294] Identification of pathogens using preserved cell cultures

[0295] Figures 17A-17D and Figure 18 show the results of a first set of experiments in which various amounts of stock cultures were loaded into the extraction chamber of the chip together with the magnetic bead solution (i.e., lysis solution and streptavidin-coated Dynal® magnetic bead suspension). The cell counts (cfu) input to the chip were from the measured stock concentration. After cell lysis and DNA capture by the beads as per the protocol above, the DNA was resuspended in 2 μL of water (without RNAse). The concentration of the extracted DNA was then measured and the master mix (assay defined above) was then added.

[0296] Amplification was then carried out in an on-chip amplification chamber.

[0297] Finally, upon amplification, the DNA concentration was measured again and the molecular count and amplification factor were derived. 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 starting DNA amount, Figure 17B - final DNA amount as a function of starting DNA amount, Figure 17C final DNA concentration as a function of starting DNA concentration, and Figure 17D - amount of extracted DNA as a function of input cell count.

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

[0299] Lane 2: DNA ladder;

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

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

[0302] lane 6: DNA ladder;

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

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

[0305] Pathogen Identification Using Cell Culture Dilution Figures 19A-19D and 20 show the results of a second set of experiments in which 1 μL of stock culture solution was serially diluted with 9 μL of water to achieve cell dilutions that induced a maximum of less than one cell. Because the chip-based approach can only accommodate very small volumes (typically a few microliters), the culture dilutions were then spun down to ensure the use of all cells within the dilution. These spun down cell pellets were then added to 10 μL of Dynal® magnetic bead solution, and an extraction process similar to the first set of experiments was performed.

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

[0307] DNA concentrations were measured before and after amplification. The data was then used to derive DNA concentrations 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 - measured number of DNA molecules as a function of input cell number, Figure 19B - final number of DNA molecules after amplification and before amplification as a function of starting number of DNA molecules, Figure 19C - amplification factor as a function of measured starting number of DNA molecules, and Figure 19D - amplification factor as a function of theoretical starting number of DNA molecules.

[0308] Using conventional laboratory-based gel electrophoresis, the size of the resulting amplification product 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 for DH10B cell amplification (approximately 700 bp);

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

[0312] lane 7: DNA ladder;

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

[0314] result

[0315] As evident from any of the above amplification experiments, there is a decrease in efficiency (i.e., as implied by the amplification factor) as the concentration of the starting sample increases. In an ideal assay where the theoretical limiting factors are negligible, exponential amplification would be expected. However, given limitations in factors such as (1) loss of enzyme efficiency after repeated cycles, (2) lack of sufficient dNTPs and primer sets, and (3) adsorption factors that inhibit recovery of amplified DNA or cause insufficient inhibition of amplified DNA (which is inversely enhanced by the on-chip surface-to-volume ratio), the theoretical amount of amplification may be difficult to achieve. However, as noted in the second set of experiments, 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 current commercially available PCR machines for viral load studies, systems according to embodiments of the present invention do not require any offline sample preparation, and the processing steps are integrated into the customized nanochip: biofluids are applied directly to the nanochip contained within the device.

[0318] Systems according to embodiments of the invention relate to amounts 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 specimen, the sooner the detection signal exceeds the baseline value. The lower the HIV-1 titer of the sample, the more number of amplification cycles will be 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 number of cycles in minutes at which the detector signal crosses a given threshold and begins the exponential growth phase. A higher Ct value indicates a lower threshold of starting HIV-1 target material.

[0319] FIG. 23 shows target standard curves for a dilution series ranging from 106 HIV-1 to 102 HIV-1 cDNA molecules in an experiment according to an embodiment of the invention. As the concentration of HIV-1 material increases, the standard curves show a shift to earlier 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 (FIG. 23). The horizontal blue lines are the Ct values ​​for this set of experiments. Pilot experiments with an embodiment according to the invention have demonstrated a working range of HIV detection from under 50 copies to 2 million copies of the starting HIV-1 cDNA or RNA molecule, as well as proof of concept with other pathogens, and experiments are underway to replicate the single molecular weight of detection already achieved with another pathogen (E. coli). In the top left corner of FIG. 23 is a picture of a prototype device according to an embodiment of the invention, smaller than a conventional laptop computer. A larger view of the device is shown in Figure 26, showing the display screen 2601 and the compartment 2602 into which the chip containing the biological sample for analysis is inserted. The device's operating system is intuitive to the user and provides seamless functionality with innovative integrated capabilities for data sharing and storage. The data in Figure 23 was generated in less than one hour.

[0320] Devices according to embodiments of the present invention have been shown to generate standard curves for quantification starting from armored HIV-1 RNA viral reference samples that contain precisely quantified copy numbers of HIV-1 RNA viral sequence targets. Armored RNA controls have become an industry standard in a wide range of diagnostic tests, including many commercially available HIV viral load tests.

[0321] Head-to-head comparison of FDA-approved Stratagene PCR machines

[0322] Further feasibility data on a system according to an embodiment of the present invention was provided by benchmarking the prototype against a gold standard quantitative PCR machine, the Stratagene MX4000.

[0323] In FIG. 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 generated by SYBR fluorescence. These standard curves were reproduced based on known HIV-1 viruses. FIG. 24 shows the operating range of a conventional PCR machine amplifying target HIV-1. The horizontal blue line reflects the Ct values ​​for this set of experiments. FIG. 23 (top) shows the same samples with known concentrations of HIV-1 with a system according to an embodiment of the invention. These data provide proof of concept that quantitative viral load data from an embodiment of the invention is essentially equivalent to the Stratagene system based on Ct, curve fitting, and operating range. Compared to a system according to an embodiment of the invention, the size of most standard FDA approved viral load Stratagene machines is approximately 10 times larger.

[0324] Detection of bacterial load with precise quantification

[0325] The common bacterial pathogen E. coli was tested on a system according to an embodiment of the invention, demonstrating an operating range of detection from 1000 copies to 10,000,000 E. coli DNA / ul (Figure 25A, left). The Pearson correlation coefficient R2 = 0.9886 indicates a statistically significant linear fit between quantification cycles and DNA copy number, demonstrating the fidelity of the technology (Figure 25B, right).

[0326] The teachings of all patents, published applications and references cited herein are hereby incorporated by reference.

[0327] Although the present invention has been particularly shown and described with reference to exemplary embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. 1. A system for rapid analysis of a biological sample, comprising: a mobile device receiving at least one integrated chip; said mobile device processes the integrated chip and analyzes the biological sample loaded thereon; the mobile device and the integrated chip together are configured to perform at least one of manipulation and control of molecules or fluidic systems on the integrated chip; And, wherein the mobile device and the integrated chip together are configured to precisely control at least one parameter governing 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%.

2. The mobile device comprises a portable control assembly that receives the at least one compact integrated chip, the integrated chip comprising: An extraction module; a nucleic acid amplification module in fluid communication with the extraction module; and a biological sample detection module in fluid communication with the nucleic acid amplification module or the extraction module; The portable control assembly includes: Extraction control module; a nucleic acid amplification control module operably linked to the extraction control module; and The system of claim 1 , further comprising a biological sample detection control module operatively linked to said nucleic acid amplification module and extraction module to process said integrated chip and analyze biological samples loaded thereon.

3. The system of claim 1 or 2, wherein the portable control assembly further comprises means for loading a biological sample, and optionally one or more reagents, onto the integrated chip.

4. The system according to any one of claims 1 to 3, wherein the nucleic acid extraction control module further comprises a chemical lysis means for carrying out lysis of cells in the biological sample loaded on the integrated chip.

5. The system according to any one of claims 1 to 4, wherein the extraction control module further comprises an ultrasonic processing means for generating ultrasonic waves within the biological sample loaded on the integrated chip, the ultrasonic waves being sufficient to disrupt cell membranes of cells of the biological sample.

6. The system according to any one of claims 1 to 5, wherein the extraction control module further comprises a fluid pressure generating means for generating a fluid pressure sufficient to mechanically shear cells of a biological sample loaded onto the integrated chip on a microfabricated protrusion disposed on the integrated chip.

7. The system according to any one of claims 1 to 6, wherein the extraction control module further comprises an electroporation means for applying a variable voltage to the biological sample loaded on the integrated chip, the voltage being sufficient to destroy cells of the biological sample.

8. The system according to any one of claims 1 to 7, wherein the extraction control module further comprises a silica particle capture means for capturing silica particles introduced into the biological sample loaded onto the integrated chip.

9. The system according to any one of claims 1 to 8, wherein the nucleic acid extraction control module further comprises a magnetic particle capture means for capturing magnetic particles introduced into a biological sample loaded onto the integrated chip.

10. The system according to any one of claims 1 to 9, wherein the magnetic particle capture means comprises a magnet.

11. The system according to any one of claims 1 to 10, wherein the nucleic acid amplification module further comprises means for well-based nucleic acid amplification.

12. The system according to any one of claims 1 to 11, wherein the means for well-based nucleic acid amplification comprises thermoelectric heating means for heating / cooling a nucleic acid amplification module of the integrated chip.

13. The system according to any one of claims 1 to 12, wherein the thermoelectric heating means for heating / cooling the nucleic acid amplification module of the integrated chip includes a temperature sensor, a power control means for controlling the thermoelectric heating means, and a processing unit for storing and executing instructions for nucleic acid amplification.

14. The system according to any one of claims 1 to 13, wherein the means for well-based nucleic acid amplification comprises means for infrared heating of the nucleic acid amplification module of the integrated chip.

15. The system according to any one of claims 1 to 14, wherein the means for well-based nucleic acid amplification comprises means for microwave heating of the nucleic acid amplification module of the integrated chip.

16. The system according to any one of claims 1 to 15, wherein the means for well-based nucleic acid amplification comprises means for applying controlled tension to nucleic acid strands within the nucleic acid amplification module of the integrated chip.

17. The system according to any one of claims 1 to 16, wherein the controlled tension is mechanical tension, hydrodynamic tension or electromagnetic tension.

18. The system of any one of claims 1 to 17, wherein the means for applying controlled tension to the nucleic acid strand is configured to operate thermostatically.

19. The system of any one of claims 1 to 18, wherein the nucleic acid amplification module further comprises a fluid-based nucleic acid amplification means comprising a fluid flow generating means for generating a flow of a buffer solution through the nucleic acid amplification module of the integrated chip, the fluid flow generating means being functionally linked to a temperature control means for controlling the temperature of the nucleic acid amplification module of the integrated chip.

20. The system according to any one of claims 1 to 19, wherein the nucleic acid amplification module comprises a real-time polymerase chain reaction (PCR) control means for carrying out nucleic acid amplification.

21. The system of any one of claims 1 to 20, wherein the biological sample detection control module comprises a fluorescent detection means for detecting nucleic acids.

22. A system according to any one of the preceding claims, wherein the fluorescence detection means comprises a light emitting diode and / or a laser diode, a data acquisition device and a processing unit for storing and processing the acquired data.

23. 23. The system of claim 22, wherein the nucleic acid detection control module further comprises a capillary electrophoresis (CE) control means for performing nucleic acid separation, the CE control means being operatively linked to the fluorescence detection means.

24. The system of any one of claims 1 to 23, wherein the CE control means further comprises a high voltage control section for applying a voltage across the nucleic acid detection module of the integrated chip, the voltage being sufficient to perform nucleic acid separation.

25. The system of any one of claims 1 to 24, wherein the nucleic acid detection control module further comprises a hybridization microarray.

26. The system according to any one of claims 1 to 25, wherein the nucleic acid detection control module comprises an electrochemical means for detecting nucleic acids.

27. The system according to any one of claims 1 to 26, wherein the nucleic acid detection control module comprises an impedance measuring means for detecting a nucleic acid.

28. The system according to any one of claims 1 to 27, further comprising a fluid pressure generating means for moving the biological sample and / or nucleic acid through the integrated chip.

29. The system according to any one of claims 1 to 28, further comprising data processing means for storing, classifying, quantifying and communicating data obtained from analysing said biological sample.

30. A system according to any preceding claim, wherein said data processing means comprises means for wireless data transmission.

31. The system according to any one of claims 1 to 30, wherein the data processing means further comprises a virus load calculation means for calculating a virus load.

32. A system according to any preceding claim, further comprising data display means for outputting data.

33. The system of any one of claims 1 to 32, wherein the system is a mobile device.

34. The system of any one of claims 1 to 33, wherein the integrated chip is disposable.

35. 1. A system for rapid analysis of a biological sample, comprising: a portable control assembly for receiving at least one compact integrated chip, the compact integrated chip comprising: an injection port for loading a biological sample onto the integrated chip, and optionally loading one or more reagents; Extraction module; a nucleic acid amplification module in fluid communication with the extraction module; a detection module in fluid communication with the nucleic acid amplification module; The portable assembly comprises: an extraction control module including a magnetic particle capture means for capturing magnetic particles introduced into the biological sample loaded on the integrated chip; a nucleic acid amplification control module operatively linked to the extraction control module, the nucleic acid amplification control module comprising a thermoelectric heating means for heating / cooling the nucleic acid amplification module of the integrated chip; and a detection control module operatively linked to the nucleic acid amplification module and the nucleic acid extraction module, comprising: A fluorescent detection means for detecting nucleic acids or proteins; and the detection control module comprising a capillary electrophoresis (CE) control means operatively linked to the fluorescence detection means, the CE control means further comprising a high voltage controller for applying a voltage sufficient to perform nucleic acid or protein separation across the nucleic acid detection module of the integrated chip; and The system further comprises a fluid pressure generating means for moving the biological sample and / or nucleic acid through the integrated chip.

36. 36. The system of claim 35, wherein the magnetic particle capture means comprises a permanent and / or electromagnet for capturing magnetic beads.

37. 37. A system according to claim 35 or 36, wherein the fluorescence detection means comprises a light emitting diode and / or a laser diode, a data acquisition device and a processing unit for storing and processing the acquired data.

38. A system according to any one of claims 35 to 37, further comprising data processing means for storing, classifying, quantifying and communicating the data.

39. A system according to any one of claims 35 to 38, wherein said data processing means comprises means for wireless data transmission.

40. A system according to any one of claims 35 to 39, further comprising data display means for outputting data.

41. The system of any one of claims 35 to 40, wherein the system is handheld.

42. The system of any one of claims 35 to 41, wherein the integrated chip is disposable.

43. 1. A method for the rapid analysis of a biological sample, comprising: (1) providing at least one integrated chip, the integrated chip comprising: Nucleic acid extraction module; a nucleic acid amplification module in fluid communication with the nucleic acid extraction module; and providing a nucleic acid detection module in fluid communication with the nucleic acid amplification module; (2) loading at least one biological sample onto the at least one integrated chip; (3) operatively coupling a portable control assembly to at least one integrated chip, said portable control assembly comprising: Nucleic acid extraction control module; a nucleic acid amplification control module operably linked to the nucleic acid extraction control module; and providing a nucleic acid detection control module operably linked 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 a biological sample loaded onto the integrated chip.

44. 44. The method of claim 43, further comprising calculating the viral load.

45. 45. The method of claim 43 or 44, wherein the biological sample is any one or more of blood, saliva, semen, urine, amniotic fluid, cerebrospinal fluid, synovial fluid, vitreous fluid, gastric fluid, nasopharyngeal aspirate or lymphatic fluid.

46. The method according to any one of claims 43 to 45, wherein the sample is a tissue sample, a water sample, an air sample, a food sample or a crop sample.

47. 47. The method of any one of claims 43 to 46, wherein the biological sample analysis detects any one or more of a water borne pathogen, an air borne pathogen, a food borne pathogen or a crop borne pathogen.

48. 1. An integrated chip for rapid sequential extraction, amplification and separation of nucleic acids from a biological sample, said integrated chip comprising: a housing having integrated therein a microfluidic channel in fluid communication with a nucleic acid extraction module, a nucleic acid amplification module, and a nucleic acid separation module; at least one sample inlet port in fluid communication with the nucleic acid extraction module for injecting a biological sample and a reagent; 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 acid, the nucleic acid amplification chamber being connected to the extraction chamber by at least one nucleic acid transport channel; and Here, the nucleic acid separation module has at least one detection channel for separating and detecting nucleic acid, and the detection channel is connected to a nucleic acid amplification chamber by at least one amplification product transport channel.

49. 49. The integrated chip of claim 48, wherein the at least one extraction chamber contains magnetic beads.

50. 50. The integrated chip of claim 48 or 49, wherein the at least one extraction chamber contains reagents for cell lysis and nucleic acid extraction.

51. The integrated chip according to any one of claims 48 to 50, wherein the nucleic acid amplification module further comprises at least one reagent addition port in fluid communication with the at least one nucleic acid transport channel.

52. The integrated chip according to any one of claims 48 to 51, wherein the nucleic acid separation module further comprises at least one sample well in fluid communication with at least one amplification product transport channel.

53. The nucleic acid separation module comprises: at least one sample waste well in fluid communication with the at least one detection channel; a buffer waste well in fluid communication with at least one detection channel; And, The integrated chip of any one of claims 48 to 52, further comprising a buffer well in fluid communication with at least one detection channel.

54. 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 a respective sample delivery channel; at least two nucleic acid amplification chambers, each connected to one of the at least two extraction chambers by a respective nucleic acid transport channel; and The integrated chip according to any one of claims 48 to 53, comprising at least two detection channels, each of which is connected to each nucleic acid amplification chamber by a respective amplification product transport channel.

55. The integrated chip according to any one of claims 48 to 54, wherein the at least two detection channels intersect.

56. The integrated chip according to any one of claims 48 to 55, further comprising at least two reagent addition ports, each of said reagent addition ports being in fluid communication with each of said nucleic acid transport channels.

57. The integrated chip according to any one of claims 48 to 56, further comprising at least two sample wells, each of said sample wells being in fluid communication with a respective amplification product transport channel.

58. The integrated chip of any one of claims 48 to 57, wherein the at least two sample wells are configured for use in the addition and / or disposal of further reagents.

59. The integrated chip of any one of claims 48 to 58, further comprising at least one sample waste well, each of said at least one sample waste well being connected to at least two detection channels by at least two sample waste channels.

60. The integrated chip of any one of claims 48 to 59, wherein the at least one sample waste well is configured for use in the addition and / or disposal of further reagents.

61. at least one buffer waste well, each in fluid communication with at least two detection channels; and The integrated chip of any one of claims 48 to 60, further comprising a buffer well in fluid communication with the at least two detection channels.

62. 62. The integrated chip of any one of claims 48 to 61, wherein the at least one buffer waste well and the buffer well are configured for use in adding and / or disposing of further reagents.

63. The integrated chip of any one of claims 48 to 62, wherein the buffer well is positioned at an intersection of at least two detection channels, and the buffer well is in fluid communication with the intersecting detection channels.

64. At least eight sample inlet ports; at least eight nucleic acid extraction chambers; at least eight nucleic acid amplification chambers; At least eight detection channels; At least eight reagent addition ports; At least eight sample wells; At least four sample waste wells; At least four buffer waste wells; and The integrated chip of any one of claims 48 to 63, comprising at least one buffer well.

65. The integrated chip according to any one of claims 48 to 64, further comprising microfabricated protrusions disposed in the at least one nucleic acid extraction chamber and / or at least one sample transport channel.

66. The integrated chip of any one of claims 48 to 65, further comprising at least two electrodes for applying a voltage across the at least one detection channel.

67. The integrated chip according to any one of claims 48 to 66, further comprising at least one nucleic acid flow control valve for controlling the flow of fluid through said at least one nucleic acid transport channel.

68. The integrated chip according to any one of claims 48 to 67, further comprising at least one amplification product control valve for controlling fluid flow through said at least one amplification product transport channel.

69. at least two nucleic acid flow control valves for controlling the flow of fluid through each nucleic acid transport channel; and The integrated chip according to any one of claims 48 to 68, further comprising at least two amplification product control valves for controlling the flow of fluid through each amplification product transport channel.

70. The integrated chip of any one of claims 48 to 69, wherein the at least two nucleic acid flow control valves and the at least two amplification product control valves are passive plugs configured for insertion into respective reagent addition ports and / or respective sample wells.

71. The integrated chip according to any one of claims 48 to 70, wherein the at least two nucleic acid flow control valves and the at least two amplification product control valves are electromagnetically controllable valves.

72. The integrated chip according to any one of claims 48 to 71, wherein the electromagnetically controllable valve is a magnetic fluid valve.

73. 73. The integrated chip of any one of claims 48 to 72, wherein the at least two sample inlet ports, the at least two reagent addition ports, the at least two sample wells, the at least four sample waste wells, the at least four buffer waste wells, and the at least one buffer well are configured to receive passive plugs.

74. The integrated chip according to any one of claims 48 to 73, wherein the at least one extraction chamber has a volume of at least 5 μL.

75. 1. A system for rapid analysis of a biological sample, comprising: A hardware system for receiving at least one integrated chip, the integrated chip comprising: an injection port for loading a biological sample onto the integrated chip, and optionally loading one or more reagents; Nucleic acid extraction module; a nucleic acid amplification module in fluid communication with said nucleic acid extraction module; a nucleic acid separation module in fluid communication with the nucleic acid amplification module; The hardware system includes: a nucleic acid extraction control module including a magnetic particle capture means for capturing magnetic particles introduced into the biological sample loaded on the integrated chip; a nucleic acid amplification control module functionally linked to the nucleic acid extraction control module, the nucleic acid amplification control module comprising a thermoelectric heating means for heating / cooling the nucleic acid amplification module of the integrated chip; and a nucleic acid detection control module operatively linked to the nucleic acid amplification module and the nucleic acid extraction module, comprising: A fluorescent detection means for detecting nucleic acids; and the nucleic acid detection control module comprising a capillary electrophoresis (CE) control means operatively linked to the fluorescence detection means, the CE control means further comprising a high voltage controller for applying a voltage sufficient to effect separation of nucleic acids across the nucleic acid separation module of the integrated chip; and The system further comprises a fluid pressure generating means for moving the biological sample and / or nucleic acid through the integrated chip.

76. 76. The system of claim 75, wherein said magnetic particle capture means comprises a permanent and / or electromagnet for capturing magnetic beads.

77. 77. A system according to claim 75 or 76, wherein the fluorescence detection means comprises a light emitting diode and / or a laser diode, a data acquisition device and a processing unit for storing and processing the acquired data.

78. A system according to any one of claims 75 to 77, further comprising data processing means for storing, classifying, quantifying and communicating the data.

79. A system according to any one of claims 75 to 78, further comprising data display means for outputting data.

80. The system of any one of claims 75 to 79, wherein a thermoelectric heating means for heating / cooling the nucleic acid amplification module of the integrated chip includes a temperature sensor, a power control means for controlling the thermoelectric heating means, and a processing unit for storing and executing instructions for nucleic acid amplification.

81. The system of any one of claims 75 to 80, wherein the nucleic acid amplification module comprises a real-time polymerase chain reaction (PCR) control means for performing nucleic acid amplification.

82. A system according to any one of claims 75 to 81, wherein the fluorescence detection means comprises a light emitting diode and / or a laser diode, a data acquisition device and a processing unit for storing and processing the acquired data.

83. The system of any one of claims 75 to 82, wherein the nucleic acid detection control module further comprises a capillary electrophoresis (CE) control means for performing nucleic acid separation, the CE control means being operatively linked to the fluorescence detection means.

84. The system of any one of claims 75 to 83, wherein the CE control means further comprises a high voltage control section for applying a voltage across the nucleic acid separation module of the integrated chip, the voltage being sufficient to perform nucleic acid separation.

85. The system according to any one of claims 75 to 84, wherein the high voltage control unit applies 10 V to 10 kV to the entire area of ​​the nucleic acid separation module of the integrated chip.

86. The system of any one of claims 75 to 85, wherein the nucleic acid detection control module further comprises a hybridization microarray.

87. A system according to any one of claims 75 to 86, wherein the fluid pressure generating means comprises a pump module providing either positive or negative pressure.

88. 1. A method for rapid analysis of a biological sample, comprising: Providing an integrated chip, the integrated chip 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 said biological sample, said extraction chamber being connected to said sample inlet port by at least one sample transport channel; A nucleic acid amplification module comprising at least one amplification chamber for amplifying a nucleic acid, the nucleic acid amplification chamber being connected to the extraction chamber by at least one nucleic acid transport channel; and providing a nucleic acid separation module comprising at least one detection channel for separating and detecting nucleic acids, said detection channel being connected to a nucleic acid amplification chamber by at least one amplification product transport channel; injecting at least one biological sample and, optionally, a reagent into at least one extraction chamber via at least one sample injection port; extracting nucleic acids from said at least one biological sample in said at least one extraction chamber by adding said at least one biological sample to magnetic beads having molecular probes attached thereto; transporting nucleic acid from said at least one extraction chamber to said at least one nucleic acid amplification chamber; amplifying the nucleic acid in the at least one nucleic acid amplification chamber by thermal polymerase chain reaction (PCR); transporting nucleic acid from the nucleic acid amplification chamber into the at least one detection channel; and The method further comprising the steps of separating the nucleic acids by size and detecting the nucleic acids in the at least one detection channel.

89. 90. The method of claim 88, wherein said step of extracting nucleic acid from said at least one biological sample in said at least one extraction chamber comprises extracting nucleic acid from a small number to a few thousand cells.

90. 90. The method of claim 88 or 89, wherein the step of extracting nucleic acids from at least one biological sample comprises lysing cells, retaining the extracted nucleic acids, and purifying the extracted nucleic acids in the at least one extraction chamber.

91. 91. The method of any one of claims 88 to 90, further comprising removing cellular debris from said at least one extraction chamber.

92. The method according to any one of claims 88 to 91, wherein the PCR is real-time quantitative PCR.

93. Detecting nucleic acids in at least one detection channel comprises: applying a voltage across at least one detection channel; and illuminating the at least one detection channel with a light source; and 93. The method of any one of claims 88 to 92, comprising detecting a fluorescent signal from said at least one detection channel.

94. 4. The integrated chip comprising: 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 a sample delivery channel; at least two nucleic acid amplification chambers, each connected to one of the at least two extraction chambers by a nucleic acid transport channel; and at least two detection channels, each connected to one of the at least two nucleic acid amplification chambers by an amplification product transport channel; The method of any one of claims 88 to 93, wherein the at least two detection channels intersect.

95. 95. The method according to any one of claims 88 to 94, further comprising injecting at least two biological samples and optionally reagents into at least two extraction chambers via at least two sample injection ports, wherein a step of detection is carried out simultaneously or sequentially to analyze each of said at least two biological samples.

96. 4. The integrated chip comprising: at least one nucleic acid flow control valve for controlling fluid flow through said at least one nucleic acid transport channel; and 96. The method of any one of claims 88 to 95, further comprising at least one amplification product control valve for controlling fluid flow through said at least one amplification product transport channel.

97. 4. The integrated chip comprising: at least one buffer waste well, each in fluid communication with at least two detection channels; and a buffer well in fluid communication with the at least two detection channels; 97. The method of any one of claims 88 to 96, wherein the at least one buffer waste well and the buffer well are configured for use in the addition and / or disposal of further reagents.

98. 98. The method of any one of claims 88 to 97, wherein the buffer well is located at an intersection of at least two detection channels, the buffer well being in fluid communication with the intersecting detection channels.

99. 99. The method of any one of claims 88 to 98, further comprising the step of loading said at least two detection channels with a sieving matrix for nucleic acid separation.

100. 100. The method of any one of claims 88-99, wherein detecting nucleic acid in the at least one detection channel comprises detecting 0.5 μL or less of PCR amplified nucleic acid.

101. 101. The method of any one of claims 88 to 100, wherein the steps of size separating and detecting nucleic acids are performed in less than 2 hours, less than 1 hour, less than 30 minutes, less than 1 minute or less than 1 second.

102. 102. The method of any one of claims 88-101, wherein said step of extracting nucleic acids from said at least one biological sample is performed within at least one of: 3 to 4 minutes; and 5 to 30 minutes.

103. 1. A valve assembly in a microfluidic device comprising: a microfluidic channel for transporting a fluid, the microfluidic channel being formed between an upper surface and a lower surface and having a longitudinal axis; a valve port in the top surface for receiving a passive plug; and a passive plug configured for insertion into the valve port; wherein a portion of the undersurface opposite the valve port is of uniform depth along a longitudinal axis of the valve assembly.

104. 1. A valve assembly in a microfluidic device comprising: a transport channel for transporting fluid; a magnetic fluid channel intersecting said transport channel; a first ferrofluid reservoir and a second ferrofluid reservoir, both connected by the ferrofluid channel; a ferrofluid in either one or both of the ferrofluid reservoirs; and The valve assembly further comprises a permanent magnet or an electromagnet for filling the ferrofluid channel with ferrofluid.

105. 1. A valve assembly in a microfluidic device comprising: Storage facility; an inflow transport channel for transporting a fluid having an output end connected to said reservoir; first and second outlet channels for transporting fluid, each having an input end connected to said reservoir; a magnetic fluid disposed in said reservoir; and a magnet for controlling the position of the magnetic fluid in the reservoir; wherein the ferrofluid in the reservoir is: the output end of the inflow channel; or the input end of the first outflow channel rather than the input end of the second outflow channel; or closing either the input end of the second channel but not the input end of the first outflow channel; Alternatively, said valve assembly is arranged so as to close neither the output end of the inflow channel nor the input ends of the first and second outflow channels.

106. 1. A valve assembly in a microfluidic device comprising: a microfluidic channel formed between the upper surface and the lower surface for transporting fluid; a valve port on said upper surface for receiving an inflatable elastic membrane; an inflatable elastic membrane configured for insertion into the valve port; and the valve assembly comprising an inlet air tube connected to the expandable elastic membrane for inflating the expandable elastic membrane, the inlet air tube allowing fluid to move through the microfluidic channel when uninflated and inhibiting fluid from moving through the microfluidic channel when inflated.

107. 107. The valve assembly of claim 106, wherein the inflatable elastic membrane and inlet air tube are embedded in the tip.

108. 108. A valve assembly according to claim 106 or 107, wherein the inflatable elastic membrane is disposed between the upper and lower surfaces.

109. 1. A valve assembly in a microfluidic device comprising: a loading well for receiving fluid; a first transport channel coupled to said loading well; a closed chamber in fluid communication with said loading well via said first transport channel; a second transport channel connected to said closed chamber; an open well in fluid communication with said closed chamber via said second transport channel; a first inflatable elastic membrane of the first transport channel that allows fluid communication between the loading well and the closed chamber when uninflated and inhibits fluid communication between the loading well and the closed chamber when inflated; a second inflatable elastic membrane in said closed chamber that expels fluid when inflated; and the valve assembly comprising a third inflatable elastic membrane of the second transport channel that allows fluid communication between the closed chamber and the open well when not inflated and prevents fluid communication between the loading well and the closed chamber when inflated.

110. 110. The valve assembly of claim 109, further comprising first, second and third air pressure input tubes coupled to the first, second and third inflatable elastic membranes, respectively.

111. 1. A method for the rapid analysis of a biological sample, comprising: (a) providing at least one integrated chip, said integrated chip comprising: Nucleic acid extraction module; a nucleic acid amplification module in fluid communication with the nucleic acid extraction module; and providing a nucleic acid detection module in fluid communication with the nucleic acid amplification module; (b) loading said at least one biological sample onto said at least one integrated chip; (c) operatively coupling a portable control assembly to the at least one integrated chip, said portable control assembly comprising: Nucleic acid extraction control module; a nucleic acid amplification control module operably linked to the nucleic acid extraction control module; and providing a nucleic acid detection control module operably linked to the nucleic acid amplification module and the nucleic acid extraction module; (d) operating the portable control assembly to perform extraction, amplification and detection of nucleic acids of the biological sample loaded onto the integrated chip; (e) determining at least one biomarker associated with the person from whom the at least one biological sample is derived based on the detection of the nucleic acid from the biological sample; and (f) based on the at least one biomarker: (i) a dosage of at least one agent to effect therapeutic treatment of a disease condition associated with said at least one biomarker; (ii) a combination of drugs that performs a therapeutic treatment of the disease condition associated with the at least one biomarker; and (iii) determining whether the person from whom the at least one biological sample is derived will respond to a drug treatment for a disease condition associated with the at least one biomarker.

112. 112. The method of claim 111, wherein the at least one biomarker is specific to a particular strain of a pathogen or disease.

113. (i) a dosage of at least one agent to effect therapeutic treatment of a disease condition associated with said at least one biomarker; and (ii) a combination of drugs that provides a therapeutic treatment for a disease condition associated with said at least one biomarker; 113. The method of claim 111 or 112, comprising determining at least one of the dosage of the at least one agent and the combination of agents based on a particular strain, the method comprising uniquely determining at least one of the dosage of the at least one agent and the combination of agents based on a particular strain.

114. 114. The method of any one of claims 111-113, further comprising delivering a dosage or combination that is uniquely determined based on the particular strain.

115. 115. The method of any one of claims 111 to 114, wherein the particular strain comprises a strain of the Human Immunodeficiency Virus (HIV).

116. 1. A method for the rapid analysis of a biological sample, comprising: (a) providing at least one integrated chip, said integrated chip comprising: 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; (b) loading said at least one biological sample onto said at least one integrated chip; (c) operatively coupling a portable control assembly to the at least one integrated chip, said portable control assembly comprising: Nucleic acid extraction control module; a nucleic acid amplification control module operably linked to the nucleic acid extraction control module; and a nucleic acid detection control module operably linked to the nucleic acid amplification module and the nucleic acid extraction module; (d) operating the portable control assembly to perform extraction, amplification and detection of nucleic acids of the biological sample loaded onto the integrated chip; (e) determining, based on the detection of nucleic acids from the biological sample, an amount of at least one biomarker in the at least one biological sample that is associated with the person from whom the at least one biological sample is derived; and (f) determining a degree of progress of treatment of a disease condition associated with said at least one biomarker in the person from whom said at least one biological sample is derived based on the amount of said at least one biomarker in said at least one biological sample.

117. 1. A method for the rapid analysis of a biological sample, comprising: (a) providing at least one integrated chip, said integrated chip comprising: 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; (b) loading said at least one biological sample onto said at least one integrated chip; (c) operatively coupling a portable control assembly to the at least one integrated chip, said portable control assembly comprising: Nucleic acid extraction control module; a nucleic acid amplification control module operably linked to the nucleic acid extraction control module; and a nucleic acid detection control module operably linked to the nucleic acid amplification module and the nucleic acid extraction module; (d) operating the portable control assembly to perform extraction, amplification and detection of nucleic acids of the biological sample loaded onto the integrated chip; (e) determining 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 (f) determining, based on the at least one biomarker, at least one of: (i) a selection of a personal care product for the person who is a source of the at least one biological sample; (ii) a delivery amount of a personal care product for the person; (iii) a cosmetic skin type of the person; and (iv) an amount of at least one cosmetic biomarker for the person in the at least one biological sample.

118. 1. A method for the rapid analysis of a biological sample, comprising: (a) providing at least one integrated chip, said integrated chip comprising: 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; (b) loading said at least one biological sample onto said at least one integrated chip; (c) operatively coupling a portable control assembly to the at least one integrated chip, said portable control assembly comprising: Nucleic acid extraction control module; a nucleic acid amplification control module operably linked to the nucleic acid extraction control module; and a nucleic acid detection control module operably linked to the nucleic acid amplification module and the nucleic acid extraction module; (d) operating the portable control assembly to perform extraction, amplification and detection of nucleic acids of the biological sample loaded onto the integrated chip; (e) determining, based on the detection of nucleic acids from the biological sample, in the at least one biological sample, an amount of at least one cosmetic biomarker associated with the person from whom the at least one biological sample is derived; and (f) determining a degree of progression of a cosmetic treatment in the person from whom the at least one biological sample is derived based on the amount of the at least one biomarker in the at least one biological sample.

119. 1. A method for the rapid analysis of a biological sample, comprising: (a) providing at least one integrated chip, said integrated chip comprising: 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; (b) loading said at least one biological sample onto said at least one integrated chip; (c) operatively coupling a portable control assembly to the at least one integrated chip, said portable control assembly comprising: Nucleic acid extraction control module; a nucleic acid amplification control module operably linked to the nucleic acid extraction control module; and a nucleic acid detection control module operably linked to the nucleic acid amplification module and the nucleic acid extraction module; (d) operating the portable control assembly to perform extraction, amplification and detection of nucleic acids of the biological sample loaded onto the integrated chip; (e) determining 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 (f) determining the degree or type of health of the person from which the at least one biological sample is derived based on the at least one biomarker.

120. 120. The method of claim 119, wherein determining the health level or type of the person comprises determining at least one of: (i) the nutritional health level of the person, or (ii) the nutritional needs of the person, and (iii) the Ayurvedic body type of the person.

121. 121. The method of claim 119 or 120, wherein determining the degree or type of health of the person comprises quantifying at least one biomarker associated with diabetes in the person.

122. 1. A method for the rapid analysis of a biological sample, comprising: (a) providing at least one integrated chip, said integrated chip comprising: 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; (b) loading said at least one biological sample onto said at least one integrated chip; (c) operatively coupling a portable control assembly to the at least one integrated chip, said portable control assembly comprising: Nucleic acid extraction control module; a nucleic acid amplification control module operably linked to the nucleic acid extraction control module; and a nucleic acid detection control module operably linked to the nucleic acid amplification module and the nucleic acid extraction module; (d) operating the portable control assembly to perform extraction, amplification and detection of nucleic acids of the biological sample loaded onto the integrated chip; (e) determining 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 (f) determining whether a person from whom the at least one biological sample is derived is a member of a subset genotype with respect to the at least one biomarker based on the at least one biomarker.

123. 123. The method of any one of claims 1 to 122, further comprising communicating, based on detection of the nucleic acid in the biological sample, (i) a dosage of at least one agent for performing a therapeutic treatment for a disease condition; and (ii) at least one of a combination of agents for performing a therapeutic treatment for the disease condition.

124. The method of any one of claims 1 to 123, wherein the nucleic acids detected from the biological sample comprise at least one of the following: nucleic acids indicative of food safety pathogens; nucleic acids indicative of agricultural diseases; nucleic acids indicative of veterinary diseases; nucleic acids from archaeological samples; nucleic acids from forensic samples; nucleic acids from water samples; nucleic acids indicative of environmental conditions; nucleic acids from customs samples; nucleic acids related to safety; nucleic acids related to biodefense; nucleic acids related to biofuels; nucleic acids related to sports participants; and nucleic acids related to human wellness.

125. 125. The method of any one of claims 1 to 124, further comprising using a communication module coupled to the handheld control assembly to at least one of transmit and receive a conditioned data signal related to the biological sample.

126. A method according to any preceding claim, comprising performing at least one of the transmitting and receiving over a network, including a wired network.

127. A method according to any preceding claim, comprising performing at least one of the transmitting and receiving over a network, including a wireless network.

128. A method according to any preceding claim, wherein the network is arranged to operate in accordance with the Bluetooth standard.

129. A method according to any preceding claim, comprising performing at least one of the transmitting and receiving over a modem.

130. The method of any one of claims 1 to 129, comprising performing at least one of the sending and receiving over a cloud network.

131. A method according to any preceding claim, comprising performing at least one of said transmitting and receiving to provide clinical healthcare remote from said portable control assembly.

132. 132. A method according to any one of claims 1 to 131, comprising performing at least one of said transmitting and receiving to provide at least one of medical care and public health services using said portable control assembly.

133. A method according to any preceding claim, comprising performing at least one of the sending and receiving over a network including a mobile device network.

134. A method according to any preceding claim, comprising performing at least one of the sending and receiving via a text messaging system.

135. The method of any one of claims 1 to 134, comprising performing at least one of the sending and receiving over at least one of a local area network, a wide area network, and the Internet.

136. The method of any one of claims 1 to 135, wherein at least one of transmitting and receiving comprises connecting with a remote genomic database storing at least one genomic profile.

137. 137. The method of any one of claims 1 to 136, wherein at least one of the sending and receiving involves a two-way information exchange of regulated data signals between a central data center and the information transfer module.

138. 138. The method of any one of claims 1 to 137, comprising using the information transfer module to: (i) receive pathogen or disease mapping information from the central data center; and (ii) transmit at least one assay result to the central data center.

139. A method according to any preceding claim, wherein at least one of the transmitting and receiving includes transmitting geographic location information to a central data centre.

140. 140. The method of any one of claims 1 to 139, wherein at least one of the sending and receiving includes receiving notification of detection of a detected pattern of a disease outbreak from a central data center from a plurality of communication modules.

141. The method of any one of claims 1 to 140, further comprising at least one of storing, transmitting and receiving a signal profile of a single reference sample using an information transmission module coupled to the portable control assembly.

142. 142. The method of any one of claims 1-141, comprising determining at least one of viral load, expected cell count per volume, and drug dosing titration based on nucleic acid detected from the biological sample.

143. 143. A system according to any preceding claim, wherein the system is configured to enable control of molecular position or orientation to within dimensions of less than 100 nm, less than 10 nm, or less than 1 nm.

144. 144. The system of any one of claims 1 to 143, wherein the system comprises at least one reaction chamber that comprises a maximum dimension 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.

145. 145. The system of any one of claims 1 to 144, wherein the system is configured to control parameters governing reactions used to analyze biological samples 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%.

146. The system of any one of claims 1 to 145, wherein the extraction module is a nucleic acid extraction module.

147. The system of any one of claims 1 to 146, wherein the extraction module is a protein extraction module.

148. The system of any one of claims 1 to 147, wherein the nucleic acid amplification module comprises an isothermal nucleic acid amplification module.

149. The system of any one of claims 1 to 148, wherein the isothermal nucleic acid amplification module comprises at least one of: a loop-mediated isothermal amplification module, a helicase-dependent amplification module, a strand displacement amplification module, a bridge amplification module, or a rolling circle amplification module.

150. The isothermal nucleic acid amplification module comprises: (a) contacting one or more template strands of a single-stranded nucleic acid with one or more oligonucleotide primers that are complementary to a portion of the one or more template strands; (b) annealing at least one of the one or more primers to said portion of the one or more template strands that is complementary to the primer; (c) contacting one or more template strands with a nucleic acid polymerase and at least four different nucleotide triphosphates; (d) extending at least one annealed primer with a nucleic acid polymerase, thereby forming one or more extension products bound to one or more template strands; (e) separating one or more extension products from one or more template strands; and (f) a module for amplifying nucleic acid by a method comprising repeating steps (a), (b), (c), (d) and (e) to amplify nucleic acid; wherein at least one of the one or more extension products of step (e) is used as a template strand in a next cycle of steps (a) to (e); wherein in the last cycle, step (e) is optional; 150. The system of any one of claims 1-149, wherein at least one of steps (b) or (d) comprises applying tension that tends to extend the nucleic acid onto the one or more template strands.

151. The system of any one of claims 1 to 150, wherein the system is handheld.

152. 1. A method for the rapid analysis of a biological sample, comprising: (1) providing at least one integrated chip, the integrated chip comprising: A protein extraction module; and providing a protein detection module in fluid communication with the protein extraction module; (2) loading at least one biological sample onto at least one integrated chip; (3) operatively coupling a portable control assembly to at least one integrated chip, said portable control assembly comprising: A protein extraction control module; and a protein detection control module operatively linked to said protein extraction module; and, (4) operating a portable control assembly to perform extraction and detection of proteins from a biological sample loaded onto the integrated chip.

153. 1. A method for the rapid analysis of a biological sample, comprising: (a) providing at least one integrated chip, said integrated chip comprising: Nucleic acid extraction module; a nucleic acid amplification module in fluid communication with the nucleic acid extraction module; and providing a nucleic acid detection module in fluid communication with the nucleic acid amplification module; (b) loading said at least one biological sample onto said at least one integrated chip; (c) operatively coupling a portable control assembly to the at least one integrated chip, said portable control assembly comprising: Nucleic acid extraction control module; a nucleic acid amplification control module operably linked to the nucleic acid extraction control module; and providing a nucleic acid detection control module operably linked to the nucleic acid amplification module and the nucleic acid extraction module; (d) operating the portable control assembly to perform extraction, amplification and detection of nucleic acids of the biological sample loaded onto the integrated chip; (e) determining at least one biomarker associated with the person from whom the at least one biological sample is derived based on the detection of the nucleic acid from the biological sample; and (f) determining at least a portion of an individualized genomic profile of the person from which the at least one biological sample is derived based on the at least one biomarker.

154. 154. The method of claim 153, wherein determining at least a portion of the individualized genomic profile comprises detecting a nucleic acid related to the wellness of the person.

155. 154. The method of claim 153, wherein determining at least a portion of the personalized genomic profile comprises detecting a nucleic acid related to sports nutrition for the person.

156. 154. The method of claim 153, wherein determining at least a portion of the individualized genomic profile comprises detecting nucleic acids associated with the person's diet.

157. 154. The method of claim 153, wherein determining at least a portion of the individualized genomic profile comprises detecting a nucleic acid related to the person's nutritional regimen.

158. 158. The method of claim 157, wherein the nucleic acid relating to nutrition for a person comprises a nucleic acid relating to a nutritional predisposition for the person.

159. 159. The method of claim 158, wherein the predisposition comprises at least one of: a nutritional allergy, a nutritional sensitivity, and a nutritional intolerance of the person.

160. 160. The method of claim 159, wherein the nucleic acids relating to human nutrition include nucleic acids relating to at least one of: gluten sensitivity, gluten intolerance, and lactose intolerance.

161. The method of claim 153, wherein determining at least a portion of the individualized genomic profile comprises detecting nucleic acids related to at least one of: an effect of a nutritional supplement on the person; a nutritional profile of the person; an effect of a cosmeceutical on the person; an effect of a medicine on the person, an effect of a food or beverage on the person.

162. 162. The method of any one of claims 153-161, wherein determining at least a portion of the individualized genomic profile comprises detecting at least one inflammatory marker.

163. 163. The method of claim 162, wherein the at least one inflammatory marker comprises at least one inflammatory marker associated with traumatic injury.

164. 163. The method of claim 162, wherein the at least one inflammatory marker comprises at least one inflammatory marker related to dietary tolerance.

165. 154. The method of claim 153, wherein determining at least a portion of the individualized genomic profile comprises detecting at least one RNA marker for at least one phenotypic expression.

166. 154. The method of claim 153, wherein determining at least a portion of the individualized genomic profile comprises detecting a nucleic acid associated with a nutritional disorder.

167. 167. The method of claim 166, wherein the nutritional disorder comprises a micronutrient deficiency.

168. 168. The method of claim 167, wherein the micronutrients include vitamins.

169. 154. The method of claim 153, wherein determining at least a portion of the individualized genomic profile comprises determining at least one probiotic or prebiotic that is deficient or altered in the person's microbiome.

170. 154. The method of claim 153, wherein determining at least a portion of the individualized genomic profile comprises detecting a nucleic acid indicative of a type or subtype of diabetes.

171. 1. A system for rapid analysis of a biological sample, comprising: (i) at least one integrated chip comprising: 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; at least one integrated chip having at least one biological sample loaded onto said integrated chip; (ii) a portable control assembly comprising: Nucleic acid extraction control module; a nucleic acid amplification control module operably linked to the nucleic acid extraction control module; and a nucleic acid detection control module operatively linked to the nucleic acid amplification module and the nucleic acid extraction module; said portable control assembly performing the extraction, amplification and detection of nucleic acids from said biological samples loaded onto said integrated chip; (iii) a genetic analyzer configured to determine at least one biomarker associated with a person from whom the at least one biological sample is derived based on detection of nucleic acids from the biological sample; (iv) the system comprising the genetic analyzer further configured to determine at least a portion of an individualized genomic profile of a person from which the at least one biological sample is derived based on the at least one biomarker.

172. 172. The system of claim 171, further comprising a distribution control unit configured to determine at least a portion of a product to distribute based at least in part on at least a portion of the individualized genomic profile.

173. 173. The system of claim 172, further comprising a dispensing activator coupled to the dispensing control and configured to dispense at least a portion of the product under control of the dispensing control.

174. 174. The system of claim 173, wherein the dispensing activator comprises at least a portion of one of: a vending machine; an automated teller machine; and a kiosk.

175. 175. The system of claim 173 or 174, wherein the dispensing activator comprises a three dimensional printer configured to print at least a portion of the product.

176. 175. The system of claim 173 or 174, wherein the dispensing activator is configured to dispense a prepackaged product.

177. 177. The system of any one of claims 172 to 176, wherein the product is at least one of: a food, a beverage, a cosmeceutical, a nutraceutical, a medicine, a herbal medicine, an alternative medicine, and a cosmetic.

178. 178. The system of any one of claims 172 to 177, wherein the distribution control unit is configured to determine at least a portion of the product to be distributed based at least in part on at least one of nutritional predispositions, nutritional disorders, and at least one probiotic or prebiotic that is deficient or altered in the microbiome.

179. 179. A system according to any one of claims 172 to 178, wherein the system for rapid analysis of biological samples is configured to enhance a consumer's experience of obtaining the product from the dispensing activator.

180. 1. A system for rapid analysis of a biological sample, comprising: (i) at least one integrated chip comprising: 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; at least one integrated chip having at least one biological sample loaded onto said integrated chip; (ii) a portable control assembly comprising: Nucleic acid extraction control module; a nucleic acid amplification control module operably linked to the nucleic acid extraction control module; and a nucleic acid detection control module operatively linked to the nucleic acid amplification module and the nucleic acid extraction module; said portable control assembly performing the extraction, amplification and detection of nucleic acids from said biological samples loaded onto said integrated chip; (iii) a genetic analyzer configured to determine at least one biomarker associated with a person from whom the at least one biological sample is derived based on detection of nucleic acids from the biological sample; The system further comprises a cardiac control unit coupled to the genetic analysis unit and configured to control the cardiac control unit based on the at least one biomarker.

181. 181. The system of claim 180, wherein the at least one biomarker includes a biomarker related to an atherosclerotic process, and the cardiac pulsatile controller is configured to reverse atherosclerosis.

182. 182. The system of claim 180 or 181, wherein the cardiac control unit comprises an enhanced external counterpulsation (EECP) therapy machine.

183. 183. The method of any one of claims 1 to 182, comprising at least one of diagnosing, classifying or monitoring a chronic disease via a nucleic acid marker.

184. 184. The method of any one of claims 1 to 183, wherein the chronic disease comprises diabetes.

185. The method of any one of claims 1 to 184, wherein the nucleic acid marker comprises an inflammation marker.

186. 186. A method according to any one of claims 1 to 185, comprising genotyping an organism, thereby determining at least one of: predisposition to a genetic disease, strain of the disease, and antibiotic resistance of the disease state.

187. 187. The method of any one of claims 1 to 186, comprising performing at least one of point-of-care detection of HIV strains, HIV viral load determination, and HIV genotyping.

188. 188. The method of any one of claims 1 to 187, comprising determining the type of viral hepatitis.

189. 189. The method of any one of claims 1 to 188, comprising identifying a cancer gene.

190. 190. The method of any one of claims 1 to 189, comprising drug monitoring.

191. 191. The method of any one of claims 1 to 190, comprising using the mobile device to at least one of monitor mother-to-child transmission of HIV or prevent mother-to-child transmission of HIV.

192. 192. A method according to any one of claims 1 to 191, comprising determining pathogens present in the patient and at least one of the susceptibility and resistance of the pathogens to at least one antibiotic or other treatment.

193. 193. A method according to any one of claims 1 to 192, comprising at least one of performing drug monitoring, using the device as a prognostic indicator of a disease, and using the device as a diagnostic and therapeutic device.

194. 194. The system of any one of claims 1 to 193, wherein the system is configured to enable control of the position or orientation of the molecule to within dimensions of less than 100 nm, less than 10 nm, or less than 1 nm.

195. 200. The system of any one of claims 1 to 194, wherein the fluidic system comprises at least one reaction chamber comprising a maximum dimension 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.

196. The system of any one of claims 1 to 195, wherein one or more modules share a common reaction chamber.

197. 200. The system of any one of claims 1 to 196, configured for delivery to a patient as an injectable, implantable, wearable or ingestible device.

198. The system of any one of claims 1 to 197, wherein the nucleic acid amplification module is configured to perform only one cycle of amplification, replication or sequencing.

199. 200. The system of claim 198, wherein only one of the cycles is isothermal.

200. 200. The system of claim 198, wherein only one of the cycles is thermal.

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