New Apparatus and Methods for Disease Detection
20200200734 ยท 2020-06-25
Assignee
Inventors
Cpc classification
B01L2300/0627
PERFORMING OPERATIONS; TRANSPORTING
G01N33/557
PHYSICS
G01N33/54373
PHYSICS
B01L2200/0647
PERFORMING OPERATIONS; TRANSPORTING
B01L3/502761
PERFORMING OPERATIONS; TRANSPORTING
International classification
G01N33/50
PHYSICS
Abstract
The invention relates to apparatus and methods for apparatus for detecting presence or monitoring profession of a disease in a biological subject, comprising a chamber in which the biological subject passes through, and at least one detection transducer placed partially or completely in the chamber; wherein information related to properties of cells in the biological subject and of cell-surrounding media is detected by the detection transducer and collected for analysis to determine whether the disease is likely to be present with the biological subject or to determine the status of the disease, thereby providing the ability to continuously determine or monitor progression of the disease.
Claims
1. An apparatus for detecting presence or monitoring progression of a disease in a biological subject, comprising a chamber in which the biological subject passes through, and at least one detection transducer placed partially or completely in the chamber; wherein information related to properties of cells in the biological subject and of cell-surrounding media is detected by the detection transducer and collected for analysis to determine whether the disease is likely to be present with the biological subject or to determine the status of the disease, thereby providing the ability to continuously determine or monitor progression of the disease.
2. The apparatus of claim 1, wherein the properties of the cells and cell-surrounding media comprise cell signaling, cell surface properties, signal pathway affecting gene replication properties and processes, signal pathway affecting gene mutation properties and processes, signal pathway affecting protein fabrication and properties, signal pathway affecting cell replications and properties, communication pathway and signaling between proteins, cells and genes, cell surface hydrophobicity properties, cell surface hydrophobicity properties, cell surface transduction properties, cell surface signal transmission properties, cell surface geometrical properties, cell surface electrical properties, cell surface ion concentration, types and distribution properties, cell inner media electrical properties, cell inner signal transmission properties, cell inner media electrical charge properties, cell inner media ion concentrations, types, and distribution properties, cellular bulk electrical properties, cellular bulk electrical properties, cell-surrounding media signal transduction properties, cell-surrounding media electrical properties, cell-surrounding media signal transmission properties, cell-surrounding media electrical charge properties, cell-surrounding media transportation properties, cell, protein, DNA, RNA, ion, and micro vesicle transportation properties in cell-surrounding media, cell, protein, DNA, RNA, ion, and micro vesicle properties in cell-surrounding media, cell-surrounding media chemical properties, cell-surrounding media bio-physical properties, cell-surrounding media bio-chemistry properties, cell to cell-surrounding media interaction properties, cell to cell-surrounding media interface properties, cell to cell-surrounding media signaling properties, cell-surrounding media ion concentrations, types, and distribution properties, cell to cell signaling properties, cell to cell communication properties, cell-to-cell interaction properties or quantum mechanical effects; and the detected information is collected for analysis to as to whether the disease is likely to be present with or within the biological subject.
3. The apparatus of claim 2, wherein the cell surface properties comprise cell surface tension, cell surface area, cell surface charge, cell surface hydrophobicity, cell surface potential, cell surface protein types and compositions, cell surface bio-chemical components, cell surface signaling properties, cell surface mutations, or cell surface biological components; and the cell to cell interaction properties comprise cell to cell affinity, cell to cell repulsion, mechanical force, electrical force, gravitational force, chemical bonding, bio-chemical interactions, geometrical matching, bio-chemical matching, chemical matching, physical matching, biological matching, or cell to cell signaling properties.
4. (canceled)
5. The apparatus of claim 3, wherein the cell to cell signaling properties comprise signaling method, signaling strength, cell surrounding media its properties to which signal is transmitted, and signaling frequency.
6. The apparatus of claim 5, wherein the cell signaling comprises cell signal type, cell signal strength, cell signal frequency, cell interactions with cell media to which cell signal is transmitted, and cell interactions with other biological entities to which signal is transmitted.
7. The apparatus of claim 1, wherein the biological subject is a blood sample, a urine sample, or a sweat sample of a mammal; and the cell surrounding media comprises blood, proteins, red blood cells, while blood cells, T cells, other cells, gene mutations, quantum mechanical effects, DNA, RNA, or other biological entities.
8. The apparatus of claim 7, wherein the cell surrounding media properties comprise a thermal, optical, acoustical, biological, chemical, physical-chemical, electro-mechanical, electro-chemical, electro-chemical-mechanical, bio-physical, bio-chemical, bio-mechanical, bio-electrical, bio-physical-chemical, bio-electro-physical, bio-electro-mechanical, bio-electro-chemical, bio-chemical-mechanical, bio-electro-physical-chemical, bio-electro-physical-mechanical, bio-electro-chemical-mechanical, physical, an electric, magnetic, electro-magnetic, or mechanical property.
9. The apparatus of claim 8, wherein the thermal property is temperature or vibrational frequency; the optical property is optical absorption, optical transmission, optical reflection, optical-electrical property, brightness, or fluorescent emission; the radiation property is radiation emission, signal triggered by radioactive material, or information probed by radioactive material; the chemical property is pH value, chemical reaction, bio-chemical reaction, bio-electro-chemical reaction, reaction speed, reaction energy, speed of reaction, oxygen concentration, oxygen consumption rate, ionic strength, catalytic behavior, chemical additives to trigger enhanced signal response, bio-chemical additives to trigger enhanced signal response, biological additives to trigger enhanced signal response, chemicals to enhance detection sensitivity, bio-chemicals to enhance detection sensitivity, biological additives to enhance detection sensitivity, or bonding strength; the physical property is density, shape, volume, or surface area; the electrical property is surface charge, surface potential, resting potential, electrical current, electrical field distribution, surface charge distribution, cell electronic properties, cell surface electronic properties, dynamic changes in electronic properties, dynamic changes in cell electronic properties, dynamic changes in cell surface electronic properties, dynamic changes in surface electronic properties, electronic properties of cell membranes, dynamic changes in electronic properties of membrane surface, dynamic changes in electronic properties of cell membranes, electrical dipole, electrical quadruple, oscillation in electrical signal, electrical current, capacitance, three-dimensional electrical or charge cloud distribution, electrical properties at telomere of DNA and chromosome, capacitance, or impedance; the biological property comprises protein, cell, genomics, quantum mechanical effects, cellular properties (which comprise chemical, physical, bio-chemical, bio-physical, and biological aspects of surrounding liquid, gas and solid of the said cell), surface shape, surface area, surface charge, surface biological property, surface chemical property, pH, electrolyte, ionic strength, resistivity, cell concentration, or biological, electrical, physical or chemical property of solution; the acoustic property is frequency, speed of acoustic waves, acoustic frequency and intensity spectrum distribution, acoustic intensity, acoustical absorption, or acoustical resonance; the mechanical property is internal pressure, hardness, flow rate, viscosity, fluid mechanical properties, shear strength, elongation strength, fracture stress, adhesion, mechanical resonance frequency, elasticity, plasticity, or compressibility.
10. The apparatus of claim 1, wherein the apparatus comprises a micro-electro-mechanical device, a semiconductor device, a micro-fluidic device, bio-chemistry machine, an immunology machine, a voltage meter, a sequencing machine, a memory unit, a logic processing unit, an optical device, imaging device, camera, viewing station, acoustic detector, piezo-electrical detector, piezo-photronic detector, piezo-electro photronic detector, electro-optical detector, electro-thermal detector, bio-electrical detector, bio-marker detector, bio-chemical detector, chemical sensor, thermal detector, ion emission detector, photo-detector, x-ray detector, radiation material detector, electrical detector, thermal recorder, or an application specific integrated circuit chip which is internally bonded to or integrated into the chamber.
11. The apparatus of claim 1, wherein the collected information is in the physical, bio-physical, bio-chemical, biological, or chemical form.
12. The apparatus of claim 11, wherein the physical form of the collected information comprises mechanical, electrical, thermal, thermodynamic, optical, and acoustical properties of the cells or cell surrounding media.
13. The apparatus of claim 1, wherein the information is collected after a probe signal is applied to the cells or cell-surrounding media and a response signal is received.
14. The apparatus of claim 13, wherein the probe signal comprises a physical, bio-physical, bio-chemical, biological, or chemical signal.
15. The apparatus of claim 14, wherein the physical signal comprises a mechanical, electrical, thermal, thermodynamic, optical, or acoustical signal.
16. The apparatus of claim 1, wherein the disease is a cancer, an inflammatory disease, diabetes, a lung disease, a heart disease, a liver disease, a gastric disease, a biliary disease, a degradation in immune system, or a cardiovascular disease.
17. The apparatus of claim 16, wherein the cancer comprises breast cancer, lung cancer, esophageal cancer, intestine cancer, cancer related to blood, liver cancer, stomach cancer, cervical cancer, ovarian cancer, rectum cancer, colon cancer, nasopharyngeal cancer, cardiac carcinoma, uterine cancer, oophoroma, pancreatic cancer, prostate cancer, brain tumor, or circulating tumor cells; the inflammatory disease comprises acne vulgaris, asthma, autoimmune diseases, autoinflammatory diseases, celiac disease, chronic prostatitis, diverticulitis, glomerulonephritis, hidradenitis suppurativa, hypersensitivities, inflammatory bowel diseases, interstitial cystitis, otitis, pelvic inflammatory disease, reperfusion injury, rheumatic fever, rheumatoid arthritis, sarcoidosis, transplant rejection, or tasculitis; the lung disease comprises asthma, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, acute bronchitis, cystic fibrosis, pneumonia, tuberculosis, pulmonary edema, acute respiratory distress syndrome, pneumoconiosis, interstitial lung disease, pulmonary embolism, or pulmonary hypertension; the diabetes comprises Type 1 diabetes, Type 2 diabetes, or gestational diabetes; the heart disease comprises coronary artery disease, enlarged heart (cardiomegaly), heart attack, irregular heart rhythm, atrial fibrillation, heart rhythm disorders, heart valve disease, sudden cardiac death, congenital heart disease, heart muscle disease (cardiomyopathy), dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, pericarditis, pericardial effusion, marfan syndrome, or heart murmurs; the liver disease comprises fascioliasis, hepatitis, alcoholic liver disease, fatty liver disease (hepatic steatosis), hereditary diseases, Gilbert's syndrome, cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, or Budd-Chiari syndrome; the gastric disease comprises gastritis, gastric polyp, gastric ulcer, benign tumor of stomach, acute gastric mucosa lesion, antral gastritis, or gastric stromal tumors; the biliary disease comprises calculus of bile duct, cholecystolithiasis, cholecystitis, cholangiectasis, cholangitis, or gallbladder polyps; the cardiovascular disease comprises coronary artery disease, peripheral arterial disease, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, endocarditis, inflammatory cardiomegaly, myocarditis, valvular heart disease, congenital heart disease, rheumatic heart disease, coronary artery disease, peripheral arterial disease, cerebrovascular disease, or renal artery stenosis.
18. The apparatus of claim 1, further comprising a sensor positioned to be partially inside the chamber and capable of detecting a property of the biological subject at the microscopic level.
19. The apparatus of claim 18, further comprising a read-out circuitry which is connected to at least one sensor and transfers data from the sensor to a recording device, and the connection between the read-out circuit and the sensor is digital, analog, optical, thermal, piezo-electrical, piezo-photronic, piezo-electrical photronic, opto-electrical, electro-thermal, opto-thermal, electric, electromagnetic, electromechanical, or mechanical.
20. (canceled)
21. The apparatus of claim 19, wherein the sensor is positioned on the interior surface of the chamber.
22. The apparatus of claim 21, wherein each sensor is independently a thermal sensor, optical sensor, acoustical sensor, biological sensor, chemical sensor, electro-mechanical sensor, electro-chemical sensor, electro-optical sensor, electro-thermal sensor, electro-chemical-mechanical sensor, bio-chemical sensor, bio-mechanical sensor, bio-optical sensor, electro-optical sensor, bio-electro-optical sensor, bio-thermal optical sensor, electro-chemical optical sensor, bio-thermal sensor, bio-physical sensor, bio-electro-mechanical sensor, bio-electro-chemical sensor, bio-electro-optical sensor, bio-electro-thermal sensor, bio-mechanical-optical sensor, bio-mechanical thermal sensor, bio-thermal-optical sensor, bio-electro-chemical-optical sensor, bio-electro-mechanical optical sensor, bio-electro-thermal-optical sensor, bio-electro-chemical-mechanical sensor, physical sensor, mechanical sensor, piezo-electrical sensor, piezo-electro photronic sensor, piezo-photronic sensor, piezo-electro optical sensor, bio-electrical sensor, bio-marker sensor, electrical sensor, magnetic sensor, electromagnetic sensor, image sensor, or radiation sensor.
23. The apparatus of claim 22, wherein the thermal sensor comprises a resistive temperature micro-sensor, a micro-thermocouple, a thermo-diode and thermo-transistor, and a surface acoustic wave (SAW) temperature sensor; the image sensor comprises a charge coupled device (CCD) or a CMOS image sensor (CIS); the radiation sensor comprises a photoconductive device, a photovoltaic device, a pyro-electrical device, or a micro-antenna; the mechanical sensor comprises a pressure micro-sensor, micro-accelerometer, flow meter, viscosity measurement tool, micro-gyrometer, or micro flow-sensor; the magnetic sensor comprises a magneto-galvanic micro-sensor, a magneto-resistive sensor, a magneto diode, or magneto-transistor; the biochemical sensor comprises a conductimetric device, a bio-marker, a bio-marker attached to a probe structure, or a potentiometric device.
24. The apparatus of claim 19, wherein at least one sensor is a probing sensor and applies a probing or disturbing signal to the biological subject; and at least another sensor, different from the probing sensor, is a detection sensor and detects a response from the biological subject upon which the probing or disturbing signal is applied.
25. (canceled)
26. The apparatus of claim 1, wherein the chamber has a length ranging from 1 micron to 50,000 microns, from 1 micron to 15,000 micron, from 1 micron to 10,000 microns, from 1.5 microns to 5,000 microns, or from 3 microns to 1,000 microns; or has a width or height ranging from 0.1 micron to 100 microns; from 0.1 micron to 25 microns, from 1 micron to 15 microns, or from 1.2 microns to 10 microns.
27. (canceled)
28. The apparatus of claim 19, comprising at least four sensors which are located on one side, two opposite sides, or four sides of the interior surface of the chamber.
29. The apparatus of claim 28, wherein the sensors are arranged in at least two arrays, and at least one array comprises two or more sensors which are apart by a distance ranging from 0.1 micron to 500 microns, from 0.1 micron to 50 microns, form 1 micron to 100 microns, from 2.5 microns to 100 microns, or from 5 microns to 250 microns.
30. (canceled)
31. (canceled)
32. (canceled)
33. (canceled)
34. (canceled)
35. The apparatus of claim 1, wherein one signal contains information related to the disease's location or where the disease is present in the source of the biological subject, or the occurrence or type of the disease; or the signal detected comprises cellular information, protein information, gene information, and any combination thereof.
36. (canceled)
37. The apparatus of claim 1, wherein the apparatus is able to detect the presence of at least two different diseases at the same time or to determine the status or progression of a disease.
38. The apparatus of claim 37, wherein the apparatus is capable of detecting at least two different types of cancer simultaneously.
39. The apparatus of claim 1, wherein the disease comprises healthy stage, non-cancer disease stage, pre-cancer stage, early stage cancer stage, or mid to late stage cancer stage, with statistically significant detection or monitoring between any of the two stages.
40. (canceled)
41. The apparatus of claim 1, wherein the apparatus is capable of detecting at least one of biological, bio-chemistry, physical and bio-physical properties of liquid media surrounding cells, proteins, and genetic components, and shift in the said properties.
42. The apparatus of claim 41, wherein the liquid media comprises blood, urine, saliva, or sweat; the biological properties comprise protein concentrations, protein types, cellular properties, quantum mechanical effects, or genetic sequence; the physical properties comprise thermal properties, mechanical properties, electrical properties, or electro-magnetic properties; and the detected properties correlate with the immune system, disease detection capability or disease killing ability, cell signaling, communications between cells, proteins, genetic components, or effectiveness and efficiency in the cell signaling and communications, or the detected properties correlate with and provide an early detection on immune system degradation, loss of ability to detect cancer, cancer killing ability, pre-cancer stage, or early stage cancer.
43. (canceled)
44. (canceled)
45. (canceled)
46. (canceled)
47. (canceled)
48. (canceled)
49. A method for detecting the presence or progression of a disease in a biological subject, comprising detecting information related to properties of cells in the biological subject and of cell-surrounding media with an apparatus of claim 1, and analyzing the collected information to determine if the likely presence or progression of the status of the disease with the biological subject.
50. (canceled)
51. The method of claim 49, wherein the properties of the cells and cell-surrounding media comprise cell signaling, cell surface properties, or cell-to-cell interaction properties; and the detected information is collected for analysis to as to whether the disease is likely to be present with or within the biological subject.
52. (canceled)
53. (canceled)
54. (canceled)
55. (canceled)
56. (canceled)
57. The method of claim 49, wherein the method is able to detect the presence of at least two different diseases at the same time or to determine the status or progression of a disease, wherein the disease comprises healthy stage, non-cancer disease stage, pre-cancer stage, early stage cancer stage, or mid to late stage cancer stage, with statistically significant detection or monitoring between any of the two stages.
58. (canceled)
59. (canceled)
60. A method for detecting the presence or progression of a disease in a biological subject, comprising measuring a biophysical property at a microscopic level of cells in the biological subject with an apparatus of claim 1, wherein information related to the measured biological property of the cells in the biological subject is detected by the detection transducer and collected for analysis to determine whether the disease is likely to be present with the biological subject or to determine the status of the disease, thereby providing the ability to continuously determine or monitor progression of the disease.
61. The method of claim 60, wherein the determination is by comparing the biophysical information of the detected biological subject with the same biological information of a confirmed disease-free or diseased biological subject.
62. The method of claim 60, wherein the biophysical property is an electric property at the microscopic level.
63. (canceled)
64. The method of claim 62, wherein the electronic property is electrical current, electric conductance, electrical resistance, capacitance, or quantum mechanical effect.
65. The method of claim 60, wherein the method is able to detect the presence of at least two different diseases at the same time or to determine the status or progression of a disease, wherein the disease comprises healthy stage, non-cancer disease stage, pre-cancer stage, early stage cancer stage, or mid to late stage cancer stage, with statistically significant detection or monitoring between any of the two stages.
66-126. (canceled)
Description
BRIEF DESCRIPTIONS OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0209] One aspect of the present invention relates to apparatus for detecting a disease in a biological subject in vivo or in vitro (e.g., human being, an organ, a tissue, or cells in a culture). Each apparatus comprises a delivery system, at least two sub-equipment units, and optionally a central control system. Each sub-equipment is capable of measuring at least a microscopic property of a biological sample. Accordingly, the apparatus of this invention can detect different parameters of the biological subject and provide accuracy, sensitivity, specificity, efficiency, non-invasiveness, practicality, conclusive, and speed in early-stage disease detection at reduced costs. In addition, the apparatus of this invention has some major advantages, such as reducing effective foot print (e.g., defined as function per unit space), reducing space for the medical devices, reducing overall cost, and providing conclusive and effective diagnosis by one device.
[0210] The delivery system can be a fluid delivery system. By the constant pressure fluid delivery system, microscopic biological subjects can be delivered onto or into one or more desired sub-equipment units of the apparatus.
[0211] As a key component of the apparatus, the micro-device should include means to perform at least the function of addressing, controlling, forcing, receiving, amplifying, or storing information from each probing address. As an example, the apparatus can further include a central control system for controlling the biological subject matter to be transported to one or more desired sub-equipment units and reading and analyzing a detected data from each sub-equipment unit. The central control system includes a controlling circuitry, an addressing unit, an amplifier circuitry, a logic processing circuitry, a memory unit, an application specific chip, a signal transmitter, a signal receiver, or a sensor.
[0212] In some embodiments, the fluid delivering system comprises a pressure generator, a pressure regulator, a throttle valve, a pressure gauge, and distributing kits. As examples of these embodiments, the pressure generator can include a motor piston system and a bin containing compressed gas; the pressure regulator (which can consist of multiple regulators) can down-regulate or up-regulate the pressure to a desired value; the pressure gauge feeds back the measured value to the throttle valve which then regulates the pressure to approach the target value.
[0213] The biological fluid to be delivered can be a sample of a biological entity to be detected for disease or something not necessarily to be detected for disease. In some embodiments, the fluid to be delivered is liquid (e.g., a blood sample or a lymph sample). The pressure regulator can be a single pressure regulator or multiple pressure regulators which are placed in succession to either down-regulate or up-regulate the pressure to a desired level, particularly when the initial pressure is either too high or too low for a single regulator to adjust to the desired level or a level that is acceptable for an end device or target.
[0214] Optionally, the apparatus includes additional features and structures to deliver a second liquid solution containing at least an enzyme, protein, oxidant, reducing agent, catalyst, radioactive component, optical emitting component, or ionic component. This second liquid solution can be added to the sample to be measured before or during sorting of the biological subject sample to be measured, or before or during the measurement (i.e., detection) of the biological subject sample, for the purposes of further enhancing the apparatus' measurement sensitivity.
[0215] In some other embodiments, the system controller includes a pre-amplifier, a lock-in amplifier, an electrical meter, a thermal meter, a switching matrix, a system bus, a nonvolatile storage device, a random access memory, a processor, or a user interface. The interface can include a sensor which can be a thermal sensor, a flow meter, an optical sensor, an acoustic detector, a current meter, an electrical sensor, a magnetic sensor, an electro-magnetic sensor, a pH meter, a hardness measurement sensor, an imaging device, a camera, a piezo-electrical sensor, a piezo-photronic sensor, a piezo-electro photronic sensor, an electro-optical sensor, an electro-thermal sensor, a bio-electrical sensor, a bio-marker sensor, a bio-chemical sensor, a chemical sensor, an ion emission sensor, a photo-detector, an x-ray sensor, a radiation material sensor, an electrical sensor, a voltage meter, a thermal sensor, a flow meter, or a piezo-meter.
[0216] In still some other embodiments, apparatus of this invention further includes a biological interface, a system controller, a system for reclaiming or treatment medical waste. The reclaiming and treatment of medical waste can be performed by the same system or two different systems.
[0217] Another aspect of this invention provides apparatus for interacting with a cell, which include a device for sending a signal to the cell and optionally receiving a response to the signal from the cell.
[0218] In some embodiments, the interaction with the cell can be probing, detecting, sorting, communicating with, treating, or modifying with a coded signal that can be a thermal, optical, acoustical, biological, chemical, electro-mechanical, electro-chemical, electro-optical, bio-electro-optical, bio-thermal optical, electro-chemical optical, electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-electro-mechanical, bio-electro-chemical, bio-electro-chemical-mechanical, electric, magnetic, electro-magnetic, physical, or mechanical signal, or a combination thereof.
[0219] In some other embodiments, the device or the sub-equipment unit contained in the apparatus can include multiple surfaces coated with one or more elements or combinations of elements, and a control system for releasing the elements. In some instances, the control system can cause release of the elements from the device surface via an energy including but not limited to thermal energy, optical energy, acoustic energy, electrical energy, electro-magnetic energy, magnetic energy, radiation energy, or mechanical energy in a controlled manner. The energy can be in the pulsed form at desired frequencies.
[0220] In some other embodiments, the device or the sub-equipment unit contained in the apparatus includes a first component for storing or releasing one element or a combination of elements onto the surface of the cell or into the cell; and a second component for controlling the release of the elements (e.g., a circuitry for controlling the release of the elements). The elements can be a biological component, a chemical compound, ions, catalysts, Ca, C, Cl, Co, Cu, H, I, Fe, Mg, Mn, N, O, P, F, K, Na, S, Zn, or a combination thereof. The signal, pulsed or constant, can be in the form of a released element or combination of elements, and it can be carried in a liquid solution, gas, or a combination thereof. In some instances, the signal can be at a frequency ranging from about 110.sup.4 Hz to about 100 MHz or ranging from about 110.sup.4 Hz to about 10 Hz, or at an oscillation concentration ranging from about 1.0 nmol/L to about 10.0 mmol/L. Also, the signal comprises the oscillation of a biological component, a chemical compound, Ca, C, Cl, Co, Cu, H, I, Fe, Mg, Mn, N, O, P, F, K, Na, S, Zn, or a combination thereof, e.g., at desired oscillating frequencies.
[0221] In some embodiments, the signal to be sent to the cell can be in the form of oscillating element, compound, or an oscillating density of a biological component, and a response to the signal from the cell is in the form of oscillating element, compound, or an oscillating density of a biological component.
[0222] In some embodiments, the device or the sub-equipment unit can be coated with a biological film, e.g., to enhance compatibility between the device and the cell.
[0223] In some other embodiments, the device or the sub-equipment unit can include components for generating a signal to be sent to the cell, receiving a response to the signal from the cell, analyzing the response, processing the response, and interfacing between the device and the cell.
[0224] Still another aspect of this invention provides devices or sub-equipment units each including a micro-filter, a shutter, a cell counter, a selector, a micro-surgical kit, a timer, and a data processing circuitry. The micro-filter can discriminate abnormal cells by a physical property (e.g., dimension, shape, or velocity), mechanical property, electric property, magnetic property, electro-magnetic, thermal property (e.g., temperature), optical property, acoustical property, biological property, chemical property, electro-chemical property, bio-chemical property, bio-electro-chemical property, bio-electro-mechanical property, or electro-mechanical property. The devices each can also include one or more micro-filters. Each of these micro-filters can be integrated with two cell counters, one of which is installed at the entrance of each filter well, while the other is installed at the exit of each filter well. The shape of the micro-filter's well is rectangle, ellipse, circle, or polygon; and the micro-filter's dimension ranges from about 0.1 m to about 500 m or from about 5 um to about 200 um. As used herein, the term dimension means the physical or feature size of the filter opening, e.g., diameter, length, width, or height. The filter can be coated with a biological or bio-compatible film, e.g., to enhance compatibility between the device and the cell.
[0225] In addition to separation of biological entity by its size and other physical features, the filter can also contain additional features and functions to perform biological entity separation via other properties, which comprise of mechanical property, electric property, magnetic property, electro-magnetic, thermal property (e.g., temperature), optical property, acoustical property, biological property, chemical property, electro-chemical property, bio-chemical property, bio-electro-chemical property, bio-electro-mechanical property, and electro-mechanical property.
[0226] In some embodiments of these devices, the shutter sandwiched by two filter membranes can be controlled by a timer (thus time shutter). The timer can be triggered by the cell counter. For instance, when a cell passes through the cell counter of the filter entrance, the clock is triggered to reset the shutter to default position, and moves at a preset speed towards the cell pathway, and the timer records the time as the cell passes through the cell counter at the exit.
[0227] Still a further aspect of this invention provides methods for fabricating a micro-device with micro-trench and probe embedded in the micro-trench's sidewalls. A micro-trench is an unclosed tunnel (see, e.g.,
[0228] In some embodiments, the method further includes coupling two devices or sub-equipment units that are thus fabricated and symmetric (i.e., a flipped mirror) to form a detecting device with channels. The entrance of each channel can be optionally bell-mouthed, e.g., such that the size of channel's opening end (the entrance) is larger than the channel's body, thereby making it easier for a cell to enter the channel. The shape of each channel's cross-section can be rectangle, ellipse, circle, or polygon. The micro-trenches of the coupled two micro-devices can be aligned by the module of alignment marks designed on the layout of the micro-device. The dimension of the micro-trench can range from about 0.1 um to about 500 um.
[0229] Alternatively, the method can also include covering the micro-trench of the micro-device with a flat panel. Such a panel can comprise or be made with silicon, SiGe, SiO.sub.2, Al.sub.2O.sub.3, quartz, low optical loss glasses, or other optical materials. Examples of other potentially suitable optical materials include acrylate polymer, AgInSbTe, synthetic alexandrite, arsenic triselenide, arsenic trisulfide, barium fluoride, CR-39, cadmium selenide, caesium cadmium chloride, calcite, calcium fluoride, chalcogenide glass, gallium phosphide, GeSbTe, germanium, germanium dioxide, glass code, hydrogen silsesquioxane, Iceland spar, liquid crystal, lithium fluoride, lumicera, METATOY, magnesium fluoride, agnesium oxide, negative index metamaterials, neutron supermirror, phosphor, picarin, poly(methyl methacrylate), polycarbonate, potassium bromide, sapphire, scotophor, spectralon, speculum metal, split-ring resonator, strontium fluoride, yttrium aluminum garnet, yttrium lithium fluoride, yttrium orthovanadate, ZBLAN, zinc selenide, and zinc sulfide.
[0230] In other embodiments, the method can further include integrating three or more sub-equipment units or devices thus fabricated to yield an enhanced device with an array of the channels.
[0231] Another aspect of this invention relates to a set of novel process flows for fabricating micro-devices (including micro-probes and micro-indentation probes) for their applications in disease detection by measuring microscopic properties of a biological sample. The micro-devices can be integrated into detection apparatus of this invention as sub-equipment units to measure one or more properties at microscopic levels. For example, a cancerous cell may have a different hardness (harder), density (denser), and elasticity than a normal cell.
[0232] Another aspect of this invention is to involve in cellular communications and regulate cellular decision or response (such as differentiation, dedifferentiation, cell division and cell death) with fabricated signals generated by the micro-devices disclosed herein. This could be further employed to detect and treat diseases.
[0233] Another aspect of the current application is that the inventive method or measured parameter in the method is a function of at least two levels F (level 1, level 2), where level 1 can be a biological entity such as protein and level 2 can be another biological entity such as genetics, where the measured signal strength of F (level 1, level 2) is greater than the sum of the signal containing only level 1 information f (level 1) and the signal containing only level 2 information f (level 2):
Signal strength of F(level 1,level 2)>signal strength of f(level 1)+signal strength of f(level 2)
[0234] The above novel feature and property can be extended to a measured parameter which is a function containing many levels F (level 1, level 2, level 3 . . . level n). One novel and unobvious feature of this innovation is that the measured signal in a parameter containing multiple biological levels is synergistically enhanced over the measured signals with each signal containing a single biological level only. With this approach, the typically weak detection signal in disease detection such as cancer detection (especially in early stage cancer detection) can be effectively enhanced or magnified, making early disease detection possible and more effective.
[0235] To further enhance measurement capabilities, multiple micro-devices can be implemented into a piece of detection apparatus as sub-equipment units employing the time of flight technique, in which at least one probing micro-device and one sensing micro-device placed at a preset, known distance. The probing micro-device can apply a signal (e.g., a voltage, a charge, an electrical field, a laser beam, a thermal pulse, a train of ions, or an acoustic wave) to the biological sample to be measured, and the detection (sensing) micro-device can measure response from or of the biological sample after the sample has traveled a known distance and a desired period of time. For instance, a probing micro-device can apply an electrical charge to a cell first, and then a detection (sensing) micro-device subsequently measures the surface charge after a desired period of time (T) has lapsed and the cell has traveled a certain distance (L).
[0236] The micro-devices or the sub-equipment units contained in the apparatus of this invention can have a wide range of designs, structures, functionalities, flexibilities, and applications due to their diverse properties, high degree of flexibilities, and ability of integration, miniaturization, and manufacturing scalability. They include, e.g., a voltage comparator, a four point probe, a calculator, a logic circuitry, a memory unit, a micro cutter, a micro hammer, a micro shield, a micro dye, a micro pin, a micro knife, a micro needle, a micro thread holder, micro tweezers, a micro laser, a micro optical absorber, a micro mirror, a micro wheeler, a micro filter, a micro chopper, a micro shredder, micro pumps, a micro absorber, a micro signal detector, a micro driller, a micro sucker, a micro tester, a micro container, a signal transmitter, a signal generator, a friction sensor, an electrical charge sensor, a temperature sensor, a hardness detector, an acoustic wave generator, an optical wave generator, a heat generator, a micro refrigerator and a charge generator.
[0237] Further, it should be noted that advancements in manufacturing technologies have now made fabrications of a wide range of micro-devices and integration of various functions onto the same device highly feasible and cost effective. The typical human cell size is about 10 microns. Using state-of-the-art integrated circuit fabrication techniques, the minimum feature size defined on a micro-device can be as small as 0.1 micron or below. Thus, it is ideal to utilize the disclosed micro-devices for biological applications.
[0238] In terms of materials for the micro-devices in the apparatus of this invention, the general principle or consideration is the material's compatibility with a biological entity. Since the time in which a micro-device is in contact with a biological sample (e.g., a cell) may vary, depending on its intended application, a different material or a different combination of materials may be used to make the micro-device. In some special cases, the materials may dissolve in a given pH in a controlled manner and thus may be selected as an appropriate material. Other considerations include cost, simplicity, ease of use and practicality. With the significant advancements in micro fabrication technologies such as integrated circuit manufacturing technology, highly integrated devices with minimum feature size as small as 0.1 micron can now be made cost-effectively and commercially. One good example is the design and fabrication of micro electro mechanical devices (MEMS), which now are being used in a wide variety of applications in the electronics industry and beyond.
[0239] Good disease (cancer and non-cancer) detection results in terms of measurement sensitivity and specificity have been obtained on multiple types of cancer tested, demonstrating validity of the apparatus of this invention for improved ability to detect diseases (e.g., cancers), particularly in their early stages. The present invention provides novel Cancer Differentiation Analysis (CDA) liquid biopsy technology. The experimental results have also shown that multiple cancer types can be detected using the disclosed apparatus, which itself is an improvement over many existing detection apparatuses.
[0240] Specifically, studies utilizing the apparatus of this invention have been carried out on multiple types of cancer and non-cancer diseases (including an inflammatory disease, diabetes, a lung disease, a heart disease, a liver disease, a gastric disease, a biliary disease, or a cardiovascular disease). In these studies, whole blood samples were used within 5 days after being obtained and/or properly transported/stored in a 0.5-20 C. refrigerated environment. The samples of the control group were obtained from healthy people confirmed by physical examinations with normal AFP and CEA values (in normal ranges).
TABLE-US-00001 TABLE 1 Data from the Test for Lung Diseases CDA CDA CDA Gender Age Age Age Mean Median STDEV Group Samples (Male %) Range Mean Median (rel. units) (rel. units) (rel. units) CDA Control 981 54 22-91 59 61 36.55 36.20 7.18 Lung 95 71 21-90 65 67 45.75 45.66 22.67 Disease Pulmonary 75 67 21-85 65 66 45.78 45.83 9.08 infection Pneumonia 14 79 22-87 61 63 44.49 45.25 9.21 Chronic 4 100 73-90 81 81 45.63 43.55 6.56 obstructive pulmonary disease Tuberculosis 2 100 65-66 66 66 53.87 53.87 11.92
TABLE-US-00002 TABLE 2 Data from Tests for Diabetes CDA CDA CDA Gender Age Age Age Mean Median STDEV Group Samples (Male %) Range Mean Median (rel. units) (rel. units) (rel. units) CDA (rel. Control 981 54 22-91 59 61 36.55 36.20 7.18 units) Diabetes 62 55 37-86 62 62 44.31 45.01 12.47 Type-2 39 49 37-86 61 62 47.08 46.45 13.34 Diabetes Unclear 23 65 43-86 63 62 39.62 41.92 9.32 types
TABLE-US-00003 TABLE 3 Data from Tests for Heart Diseases CDA CDA CDA Gender Age Age Age Mean Median STDEV Group Samples (Male %) Range Mean Median (rel. units) (rel. units) (rel. units) CDA (rel. Control 981 54 22-91 59 61 36.55 36.20 7.18 units) Heart 54 45 21-105 73 75 44.24 44.43 11.97 Disease Coronary 26 38 50-94 71 70 41.99 42.70 13.39 disease Other 14 57 61-91 76 76 46.88 47.73 6.86 heart disease Heart failure 9 44 74-105 82 80 48.60 45.41 14.58 Arrhythmia 5 20 21-85 62 70 40.69 44.18 9.11
TABLE-US-00004 TABLE 4 Data from Tests for Liver Diseases CDA CDA CDA Gender Age Age Age Mean Median STDEV Group Samples (Male %) Range Mean Median (rel. units) (rel. units) (rel. units) CDA (rel. Control 981 54 22-91 59 61 36.55 36.20 7.18 units) Liver 160 68 24-87 55.56 53.50 44.29 44.75 8.32 Disease Cirrhosis 88 78 30-87 57.68 55.00 43.68 43.72 8.62 Hepatitis 56 63 24-76 54.27 52.50 43.32 43.84 7.74
TABLE-US-00005 TABLE 5 Data from Tests for Gastric Diseases CDA CDA CDA Gender Age Age Age Mean Median STDEV Group Samples (Male %) Range Mean Median (rel. units) (rel. units) (rel. units) CDA (rel. Control 981 54 22-91 59 61 36.55 36.20 7.18 units) Gastric 47 60 29-89 60.81 63.00 44.24 44.90 9.29 Disease Gastritis 28 61 29-89 60.29 62.00 45.16 45.01 9.37 Gastric 12 67 33-71 61.00 66.00 41.70 44.37 8.17 polyp Gastric 2 50 59-79 69.00 69.00 36.76 36.76 11.12 ulcer
TABLE-US-00006 TABLE 6 Summary of Descriptive Statistics CDA CDA CDA Gender Age Age Age Mean Median STDEV Group Samples (Male %) Range Mean Median (rel. units) (rel. units) (rel. units) CDA (rel. Control 981 54 22-91 59 61 36.55 36.20 7.18 units) Lung 95 71 21-90 65 67 45.75 45.66 22.67 Disease Diabetes 62 55 37-86 62 62 44.31 45.01 12.47 Heart 54 45 21-105 73 75 44.24 44.43 11.97 Disease Liver 160 68 24-87 55.56 53.50 44.29 44.75 8.32 Disease Gastric 47 60 29-89 60.81 63.00 44.24 44.90 9.29 Disease Biliary 28 57 21-85 60.11 60.50 45.75 46.57 11.82 Disease
TABLE-US-00007 TABLE 7 Results of ROC Curve Analysis Area Under the Curve Cut-off Value Group (rel. units) (rel. units) Sensitivity Specificity Lung Disease 0.788 41 74.7% 73.9% Diabetes 0.727 41 72.6% 72.3% Heart Disease 0.736 41 74.1% 74.3% Liver Disease 0.758 41 70.0% 73.8% Gastric Disease 0.740 41 74.5% 74.3% Biliary Disease 0.779 41 82.1% 74.4%
[0241] CDA value is obtained from an algorithm using calculation based on tested values from the studies. CDA value increases with risks of diseases. In other words, the higher the CDA values, the higher the risks of diseases.
[0242] As the above tables show, the CDA values are higher for various diseases (mid 40s) than those of control (healthy) group (around 36). Statistical analysis of CDA values for those two groups shows that there was a statistically significant difference in CDA values between those two groups. Accordingly, the studies above show that the apparatus and methods of this invention were able to distinguish some major diseases from control group, with sensitivity and specificity likely higher than existing technologies.
[0243] Set forth below are several illustrations or examples of apparatus of this invention containing a class of innovative micro-devices that are integrated as sub-equipment units.
[0244]
[0245]
[0246]
[0247] To enhance detection speed and sensitivity, a large number of micro-devices can be integrated into a single apparatus of this invention. Each micro-device can be a independent sub-equipment unit in the apparatus. To achieve the above requirements, the detection apparatus should be optimized with its surface area maximized to contact the biological sample and with large number of micro-devices integrated on the maximized surface.
[0248] Instead of measuring a single property of a biological subject for disease diagnosis, various micro-devices can be integrated into a detection apparatus to detect multiple properties. Various micro-devices can constitute different sub-equipment units.
[0249] As illustrated herein, it is desirable to optimize the detection apparatus design to maximize measurement surface area, since the greater the surface area, the greater number of micro-devices that can be placed on the detection apparatus to simultaneously measure the sample, thereby increasing detection speed and also minimizing the amount of sample needed for the test.
[0250]
[0251] Yet another aspect of this invention relates to a set of novel fabrication process flows for making micro-devices or sub-equipment units for disease detection purposes. Thus, a micro-device with two probes capable of measuring a range of properties (including mechanical and electrical properties) of biological samples is fabricated, using the above novel fabrication process flow.
[0252] Detection apparatus integrated with micro-devices disclosed in this application is fully capable of detecting pre-chosen properties on a single cell, a single DNA, a single RNA, or an individual, small sized biological matter level. In another further aspect, the invention provides the design, integration, and fabrication process flow of micro-devices capable of making highly sensitive and advanced measurements on very weak signals in biological systems for disease detection under complicated environment with very weak signal and relatively high noise background. Those novel capabilities using the class of micro-devices disclosed in this invention for disease detection include but not limited to making dynamic measurements, real time measurements (such as time of flight measurements, and combination of using probe signal and detecting response signal), phase lock-in technique to reduce background noise, and 4-point probe techniques to measure very weak signals, and unique and novel probes to measure various electronic, electromagnetic and magnetic properties of biological samples at the single cell (e.g., a telomere of DNA or chromosome), single molecule (e.g., DNA, RNA, or protein), single biological subject (e.g., virus) level.
[0253] For example, in a time of flight approach to obtain dynamic information on the biological sample (e.g., a cell, a substructure of a cell, a DNA, a RNA, or a virus), a first micro-device is first used to send a signal to perturb the biological subject to be diagnosed, and then a second micro-device is employed to accurately measure the response from the biological subject. In one embodiment, the first micro-device and the second micro-device are positioned with a desired or pre-determined distance L apart, with a biological subject to be measured flowing from the first micro-device towards the second micro-device. When the biological subject passes the first micro-device, the first micro-device sends a signal to the passing biological subject, and then the second micro-device detects the response or retention of the perturbation signal on the biological subject. From the distance between the two micro-devices, time interval, the nature of perturbation by the first micro-device, and measured changes on the biological subject during the time of flight, microscopic and dynamic properties of the biological subject can be obtained. In another embodiment, a first micro-device is used to probe the biological subject by applying a signal (e.g., an electronic charge) and the response from the biological subject is detected by a second micro-device as a function of time.
[0254] To further increase detection sensitivity, a novel detection process for disease detection is used, in which time of flight technique is employed.
[0255] The utilization of micro-devices (e.g., made by using the fabrication process flows of this invention) as discussed above and illustrated in
[0256]
[0257] In addition to the above examples in measuring electrical properties (e.g., charge, electronic states, electronic charge, electronic cloud distribution, electrical field, current, and electrical potential, and impedance), mechanical properties (e.g., hardness, density, shear strength, and fracture strength) and chemical properties (e.g., pH) in a single cell, and in
[0258]
[0259] One of the key aspects of this invention is the design and fabrication process flows of micro-devices and methods of use the micro-devices for catching and/or measuring biological subjects (e.g., cells, cell substructures, DNA, and RNA) at microscopic levels and in three dimensional space, in which the micro-devices have micro-probes arranged in three dimensional manner with feature sizes as small as a cell, DNA, or RNA, and capable of trapping, sorting, probing, measuring, and modifying biological subjects. Such micro-devices can be fabricated using state-of-the-art microelectronics processing techniques such as those used in fabricating integrated circuits. Using thin film deposition technologies such as molecular epitaxy beam (MEB) and atomic layer deposition (ALD), film thickness as thin as a few monolayers can be achieved (e.g., 4 A to 10 A). Further, using electron beam or x-ray lithography, device feature size on the order of nanometers can be obtained, making micro-device capable of trapping, probing, measuring, and modifying a biological subject (e.g., a single cell, a single DNA or RNA molecule) possible.
[0260] Another aspect of this invention relates to micro-indentation probes and micro-probes for measuring a range of physical properties (such as mechanical properties) of biological subjects. Examples of the mechanical properties include hardness, shear strength, elongation strength, fracture stress, and other properties related to cell membrane which is believed to be a critical component in disease diagnosis.
[0261] Another novel approach provided by this invention is the use of phase lock-in measurement for disease detection, which reduces background noise and effectively enhances signal to noise ratio. Generally, in this measurement approach, a periodic signal is used to probe the biological sample and response coherent to the frequency of this periodic probe signal is detected and amplified, while other signals not coherent to the frequency of the probe signal is filtered out, which thereby effectively reduces background noise. In one of the embodiments in this invention, a probing micro-device can send a periodic probe signal (e.g., a pulsed laser team, a pulsed thermal wave, or an alternating electrical field) to a biological subject, response to the probe signal by the biological subject can be detected by a detecting micro-device. The phase lock-in technique can be used to filter out unwanted noise and enhance the response signal which is synchronized to the frequency of the probe signal. The following two examples illustrate the novel features of time of flight detection arrangement in combination with phase lock-in detection technique to enhance weak signal and therefore detection sensitivity in disease detection measurements.
[0262]
[0263]
[0264]
[0265] To illustrate how a micro-device can be used to simulate an intracellular signal, calcium oscillation is taken as an example mechanism. First, a Ca.sup.2+-release-activated channel (CRAC) has to be opened to its maximal extent, which could be achieved by various approaches. In an example of the applicable approaches, a biochemical material (e.g., thapsigargin) stored in the cartridge 924 is released by an injector 925 to the cell, and the CRAC will open at the stimulus of the biological subject. In another example of the applicable approaches, the injector 924 forces a specific voltage on cell membrane, which causes the CRAC to open as well.
[0266] The Ca.sup.2+ concentration of a solution in the injector 928 can be regulated as it is a desirable combination of a Ca.sup.2+-containing solution 926, and a Ca free solution 927. While the injector 930 contains a Ca.sup.2+ free solution, then injectors 928 and 930 are alternately switched on and off at a desired frequency. As such, the Ca oscillation is achieved and the content inside the cell membrane are then exposed to a Ca oscillation. Consequently, the cell's activities or fate is being manipulated by the regulated signal generated by the apparatus.
[0267] Meanwhile, the cell's response (e.g., in the form of a thermal, optical, acoustical, mechanical, electrical, magnetic, electromagnetic property, or a combination thereof) can be monitored and recorded by the probes integrated in this apparatus.
[0268]
[0269] As surface charge will affect the shape of a biological subject, by using novel and multiple plates, information on the shape and charge distribution of biological subjects can be obtained. The general principle and design of the micro-device can be extended to a broader scope, thereby making it possible to obtain other information on the biological subject via separation by applying other parameters such as ion gradient, thermal gradient, optical beam, or another form of energy.
[0270]
[0271] Alternatively, a probe 1020 can be designed to trigger optical emission such as florescence light emission 1043 in the targeted biological subject such as diseased cells, which can then be detected by an optical probe 1032 as illustrated in
[0272]
[0273] The channel included in the apparatus of this invention can have a width of, e.g., from 1 nm to 1 mm. The apparatus should have at least one inlet channel and at least two outlet channels.
[0274]
[0275]
[0276]
[0277]
[0278]
[0279]
[0280]
[0281]
[0282]
[0283]
[0284]
[0285]
[0286]
[0287]
[0288] The biological subject 2501 flows in the x direction from the entrance channel 2510 to the accelerating chamber 2530. A bio-compatible fluid 2502 flows from disturbing fluid channel 2520 to the accelerating chamber 2530, it then accelerates the biological subject 2501 in the y-direction. The acceleration correlates with the radius of the biological subject and the larger ones are less accelerated than the smaller ones. Then, the larger and smaller subjects are separated into different selecting channels. Meanwhile, probes can be optionally assembled on the sidewalls of the channels 2510, 2520, 2530, 2540, and 2550. The probes could detect, at the microscopic level, electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, biochemical, electro-mechanical, electro-chemical, electro-chemical-mechanical, physical, mechanical properties, or combinations thereof.
[0289]
[0290]
[0291]
[0292]
[0293]
[0294]
[0295]
[0296] Probe 3112 is a fine probing device which is coated by a piezo-electrical material. There is a distance L between probe 3111 and probe 3112.
[0297] When the biological subjects are tested when getting through 3111, if the entity is identified to be a suspected abnormal one, the system would trigger the piezo-electrical probe 3112 to stretch into the channel and probe particular properties after a time delay of t. And probe 3112 retracts after the suspected entity passed through.
[0298] The probing device is capable of measuring at the microscopic level an electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, electro-mechanical, electro-chemical, electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-electro-mechanical, bio-electro-chemical, bio-electro-chemical-mechanical, physical or mechanical property, or a combination thereof, of the biological subject.
[0299] The width of the micro-channel can range from about 1 nm to about 1 mm.
[0300]
[0301] When a biological subject is tested while getting through 3211, if it is normal, the valve 3221 of the flush channel is open, while the detection channel valve 3222 is closed, the biological subject is flushed out without a time-consuming fine detection.
[0302] When the biological subject is tested while getting through 3211, if it is suspected to be abnormal or diseased, the valve 3221 of the flush channel is closed, while the detection channel valve 3222 is open, the biological subject is conducted to the detection channel for a more particular probing.
[0303] The width of the micro-channel can range from about 1 nm to about 1 mm.
[0304] The probing device is capable of measuring at the microscopic level an electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, electro-mechanical, electro-chemical, electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-electro-mechanical, bio-electro-chemical, bio-electro-chemical-mechanical, physical or mechanical property, or a combination thereof, of the biological subject.
[0305]
[0306] The probing device is capable of measuring at the microscopic level an electrical, magnetic, electromagnetic, thermal, optical, acoustical, biological, chemical, electro-mechanical, electro-chemical, electro-chemical-mechanical, bio-chemical, bio-mechanical, bio-electro-mechanical, bio-electro-chemical, bio-electro-chemical-mechanical, physical or mechanical property, or a combination thereof, of the biological subject.
[0307]
[0308] As illustrated in
[0309]
[0310]
[0311]
[0312]
[0313]
[0314]
[0315]
[0316] In
[0317]
[0318]
[0319] Like
[0320] To effectively sorting, separating, screening, probing, or detecting of diseased biological entities, a chamber (or chambers) integrated with various channels can be deployed as shown
[0321] To significantly speed up the sorting, screening, probing and detection operations using the disclosed device and process, a high number of desired structures such as those discussed in
[0322]
[0323]
[0324]
[0325] One of the key aspects of the present invention relates to a novel technology for detecting disease, in which a number of different classifications of biological information are collected in a device and processed or analyzed. For instance,
[0326] Tests were carried out in the laboratory with the apparatus of this invention on certain cancerous tissue samples (with multiple samples for each type of cancer) although the apparatus of this invention can be used for detection of other types of cancer or other types of treatment. In the tests, healthy control samples were obtained from animals with no known cancer disease at the time of collection and no history of malignant disease. Both cancerous samples and healthy control samples were collected and cultured in the same type of culture solution. The cultured samples were then mixed with a dilution buffer and diluted to the same concentration. The diluted samples were maintained at the room temperature for different time intervals and processed within a maximum of 6 hours after being recovered. The diluted samples were tested at the room temperature (2023 C.) and in the humidity of 30%40%. The samples were tested with an apparatus of this invention under the same conditions and stimulated by the same pulse signal.
[0327] The tests show that, in general, the control groups' tested (measured) values (i.e., measured values in relative units for the testing parameter) were lower than the cancerous or diseased groups. Under the same stimulation (in terms of stimulation type and level) with a stimulating or probing signal applied by a probing unit of the tested apparatus of this invention, the difference shown in the measured values between the control groups and the cancerous groups became much more significant, e.g., ranging from 1.5 times to almost 8 times in terms of level of increase in such difference, compared with that without simulation. In other words, the cancerous groups' response to the stimulating signal was much higher than that of the control groups. Thus, the apparatus of this invention has been proven to be able to significantly enhance the relative sensitivity and specificity in the detection and measurement of diseased cells, in comparison to the control or healthy cells.
[0328] Further, the test results show that in terms of the novel parameter utilized by the apparatus of this invention, the cancerous group and the control group showed significantly different response. Such difference is significantly greater than the measurement noise. There was a large window to separate the control groups from the cancerous groups, showing a high degree of sensitivity of the novel measurement method and apparatus.
[0329]
TABLE-US-00008 Traditional technology This invention P - protein based (bio-marker, AFP, CEA, PSA, etc.) P C - cellular based (CTC, ctDNA) + C M - Molecular (genomics, DNA, RNA) + M => one-dimensional information + P-C + P-M + M-C + M-C-P => seven dimensional info Other level/parameter (O) + M-C-P-O => more dimensions info
[0330]
[0331]
[0332]
[0333] Studies were also undertaken to examine the effect of adding molecular level reaction triggering agent on the efficacy of the apparatus and methods for detecting disease of this invention. The results provided in
[0334] The apparatus and methods of this invention has been used in test of more than 20 different types of cancer in all stages of development and showed expectedly high sensitivity and specificity.
[0335]
[0336]
[0337]
[0338] Another major novel aspect of this application relates to an effective method to probe and track ability (including immune system) to detect and prevent potential diseases, ability to flight diseases, and the state of a life body, including but not limited to healthy state, non-cancer disease state, pre-cancer state, and cancer state.
[0339] Using a novel microfluidic device equipped with sensitive sensors and a fully automated testing machine developed in this work, the method of this invention has been demonstrated on about 100,000 samples which included control (healthy group), disease group, pre-cancer disease group, and cancer group individuals. The test results showed statistically significant blood micro-electrical current level decreasing from healthy group to disease group, and further decreasing to cancer group, signifying potential importance of this new detection technology for early stage cancer detection. In early stage non-small cell lung cancer (NSCLC) tests, sensitivity and specificity reached 85% and 93%, respectively. It has also shown that it is capable to detect over 20 types of cancer, including esophageal cancer and brain tumor which do not have other effective screening methods. As the class of electrical properties is a fundamental bio-physical sub-field and impacting many aspects of human blood, it has multi-level effects at cellular, protein, and even molecular levels. Data appear to reveal that this novel technology provides a potentially powerful insight into how cancer evolves and can be highly valuable for pre-cancer and early stage cancer detection. Its mechanism, potential significance, and ramifications will be presented.
[0340] Since the liquid media (for example, blood) is interfacing, connecting and communicating with both cells, proteins, and genetic components (DNAs, RNAs, etc.), it plays a critical role in the interfacing, interactions, and communications (for example, cell signaling) between cells, proteins, and genetic components (DNAs, RNAs, etc.) and other biological entities, and the occurrence and progression of diseases including but not limited to non-cancer diseases, pre-cancer diseases and cancer. On the other hand, in the transition from a healthy individual to a disease state, immune system is degraded and disease detection and killing agents such as T cell lost function. In this invention, it is believed that immune system degradation (decrease) and loss in disease detection and disease fighting will and action is caused by changes in properties in the said liquid media surrounding cells, proteins, genetic components (DNAs, RNAs, etc.) and other biological entities. Specifically, those properties can be biological properties (protein concentration, protein types, DNA sequence, DNA static electrical force, DNA surface charge, DNA surrounding media electrical properties, quantum mechanical effects, etc.), bio-chemistry properties, physical properties (thermal, mechanical, electrical, and electro-magnetic properties), bio-physical properties, properties. For example, the shift in the above property (for example, reduction in the above said physical properties) may affect (for example, reduction in effectiveness and efficiency, and transduction degradation) cell signaling and communications by cells and between cells and other biological entities, resulting in the compromise of immune system, loss of detection capability of cells such as T cells to detect cancer cells and ability to kill cancer cells. Therefore, by measuring the above properties including physical and bio-physical properties, one is able to detect the onset of disease and track disease from one stage to the next stage, making early detection and prevention of disease possible.
[0341]
Exemplary Test
[0342] Mechanism
[0343] A micro fluidic device was fabricated by an integrated circuit method in which micro-channels were formed along which sample fluid can be passed, and on whose sides detection transducers (i.e., sensors) were formed to probe the fluid. During dada collection, a voltage meter with automated data recording capabilities was used. When fluid sample arrives at a micro-channel, sensors in the channel can probe the sample via applying a constant voltage while recording micro-electrical current response as a function of time dependent behavior (time sweep) as shown
[0344] Cell Line Characteristics
[0345] Four cell lines were utilized in the preliminary research. Human non-small cell lung cancer cell line A-549 (Cat. No. TCHu150), human embryonic lung cell line MRC-5 (Cat. No. GNHu41), human hepatoma cell line QGY (Cat. No. TCHu 42) and human hepatocyte cell line HL-7702 (Cat. No. GNHu 6), which were purchased from Cell Bank of Typical Culture Preservation Committee of Chinese Academy of Sciences/Cell Resource Center of Shanghai Academy of Life Sciences, Chinese Academy of Sciences, were cultured in complete growth medium of RPMI-1640 medium which contain 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin in atmosphere of 95% air and 5% carbon dioxide in 37 C. Cell suspension solutions were prepared for testing.
[0346] Blood Sample Characteristics
[0347] Samples used in a CDA test were whole blood or serum samples, with whole blood typically used.
[0348] Whole blood was drawn into an EDTA tube with anticoagulant agent. In addition, cell lines for both control (healthy) and cancer samples were also used in initial development phase of the work to test and validate signals of the technology.
[0349] Algorithm
[0350] With a large data base from retrospective studies, an algorithm has been built with a CVD test numbers along with cut-off values as a test outcome which is correlated to cancer risk, which (CDA value) is proportional to cancer risk. Based on CDA values, three regions were divided, healthy, medium risk, and high risk.
[0351] Results
[0352] Both retrospective studies and population screenings were carried out. For both medium risk and high risk groups, a follow-up was carried out on randomly selected 3,000 individuals. For the 3,000 individuals, feedback on 2,000 was obtained.
[0353]
[0354] Furthermore, there is noticeable difference between control, disease and liver cancer samples (
[0355]
[0356] Data for a typical control whole blood sample and a liver cancer whole blood sample are shown in
[0357]
[0358] Having initially confirmed feasibility of this new technology for disease detection, multiple retrospective clinical studies have been carried out. Data on over 20 types of cancer have been collected, and an algorithm has been built based upon a large data base. A set of test parameters have been built around the above-mentioned algorithm. The key parameter calculated from this algorithm based on raw data is CDA indicator, whose value is proportional to the cancer risk, and inversely proportional micro-electrical current value of the sample tested.
[0359] Table 8 shows significance test of differencenon-parametric test of various types of cancer. In Table 8, the distribution of CDA is the same across the categories of Group. Asymptotic significances are displayed. The significance level is 0.05. Table 8 shows that the difference in CDA values between control group and various cancer types are of statistical significance.
TABLE-US-00009 TABLE 8 Hypothesis Test Summary Null Hypothesis Test Sig. Decision Control (1717) vs. Independent 0.000 Reject the Cancer (10078) Samples null Hypothesis Control (1717) vs. Mann Whitney 0.000 Reject the Lung Cancer (1907) U Test null Hypothesis Control (1717) vs. 0.000 Reject the Colon Cancer (710) null Hypothesis Control (1717) vs. 0.000 Reject the Esophageal Cancer (1590) null Hypothesis Control (1717) vs. 0.000 Reject the Gastric Cancer (1H7) null Hypothesis Control (1717) vs. 0.000 Reject the Rectal Cancer (522) null Hypothesis Control (1717) vs. 0.000 Reject the Cardia Cancer (135) null Hypothesis Control (1717) vs. 0.000 Reject the Liver Cancer (738) null Hypothesis Control (1717) vs. 0.000 Reject the Pancreatic Cancer (134) null Hypothesis Control (1717) vs. 0.000 Reject the Ovarian Cancer (337) null Hypothesis Control (1717) vs. 0.000 Reject the Breast Cancer (348) null Hypothesis Control (1717) vs. 0.000 Reject the Cervical Cancer (318) null Hypothesis Control (1717) vs. 0.000 Reject the Uterine Cancer (105) null Hypothesis Control (1717) vs. 0.000 Reject the Prostatic Cancer (31) null Hypothesis Control (1717) vs. 0.000 Reject the Brain Tumor (50) null Hypothesis Control (1717) vs. 0.000 Reject the Lymphoma (322) null Hypothesis Control (1717) vs. 0.000 Reject the Nasopharyngeal null Hypothesis Cancer (121) Control (1717) vs. 0.000 Reject the Other Cancer (1593) null Hypothesis
[0360] A summary of cancer screening sensitivity and specificity for control group and a number of cancer types from retrospective study is given in Table 9. Table 9 showed that overall, both sensitivity and specificity of CDA technology of various cancer types are relatively high, demonstrating CDA technology is potentially suited for a large number of cancer types. In addition, statistical analysis of the data Table 8 showed that P values for each two groups (each cancer group and control group) are all less than 0.001, also meaning that the difference in CDA values between control group and various cancer types listed in Table 8 are of statistical significance.
TABLE-US-00010 TABLE 9 CDA technology demonstrates high sensitivity and specificity for cancer screening of various types of cancer Control (1717) vs. Sensitivity Specificity Cancer (10078) 86.6% 86.9% Lung Cancer (1907) 88.4% 88.4% Colon Cancer (710) 87.7% 87.4% Esophageal Cancer (1590) 86.9% 86.8% Gastric Cancer (1117) 82.4% 86.8% Rectal Cancer (522) 83.1% 86.8% Cardia Cancer (135) 79.3% 87.0% Liver Cancer (738) 89.7% 88.8% Pancreatic Cancer (134) 82.8% 88.0% Ovarian (337) 85.5% 86.9% Breast Cancer (348) 86.2% 87.1% Cervical Cancer (318) 84.0% 87.4% Uterine Cancer (105) 84.8% 87.3% Prostatic Cancer (31) 80.6% 87.5% Brain Tumor (50) 82.0% 87.1% Lymphoma (322) 87.6% 87.7% Nasopharyngeal Cancer (121) 81.0% 87.1% Other cancer (1593) 85.6% 86.8%
[0361] Table 10 shows CDA values of non-small lung cancer samples at various stages and control sample, and corresponding sensitivity and specificity, which are higher than traditional methods, particularly at stage I.
TABLE-US-00011 TABLE 10 CDA technology demonstrates high sensitivity and specificity for early stage screening of NSCLC Average Median SD of Sample CDA (rel. CDA (rel. CDA (rel. Group Size units) units) units) Sensitivity Specificity Control 248 33.98 34.72 5.50 / / NSCLC Stage I 108 49.49 50.63 9.03 85.2% 90.7% Stage II 90 52.38 53.66 7.21 93.3% 91.1% Stage III 246 53.66 53.87 5.26 98.0% 95.6% Stage IV 388 52.45 52.96 6.11 95.1% 95.2%
[0362] Esophageal cancer is a cancer which still does not have a bio-marker and IVD screening method. In this investigation, CDA technology has been evaluated for esophageal cancer screening. Esophageal cancer results are summarized in Table 11. Results showed even at stage I, sensitivity and specificity are above 80%, far better than those by other technologies, which will have significant clinical meaning in catching esophageal cancer early.
TABLE-US-00012 TABLE 11 CDA technology demonstrates high sensitivity and specificity for early stage screening of esophageal cancer Average Median SD of Sample CDA (rel. CDA (rel. CDA (rel. Group Size units) units) units) Sensitivity Specificity Control 248 33.98 34.72 5.50 / / Esophageal Stage I 38 47.38 48.47 6.78 81.6% 84.7% Cancer Stage II 88 45.63 44.96 10.28 80.7% 84.7% Stage III 95 47.37 46.03 9.66 80.0% 84.7% Stage IV 63 54.37 53.22 16.04 85.7% 85.1%
[0363] CDA technology was utilized to screen 70,000 general populations. Based on CDA values, screened individuals were divided into three groups: low risk, medium risk, and high risk. Follow-up was carried out on about 3600 individuals with medium to high risk values, out of which 2240 individuals were able to have made contact and willing to share results from follow-up tests and diagnosis. Table 12A shows cancer cases screened out by CDA technology (based on follow-up on 2240 individuals initially tested with medium and high CDA values and later confirmed by oncologists). Table 12B shows pre-cancer cases screened out by CDA technology (based on follow-up on 2240 individuals initially tested with medium and high CDA values and later confirmed by oncologists). As shown in Table 12A and Table 12B, at the time of the follow-up contact, 73 individuals were diagnosed by oncologists having cancer, and 113 individuals were confirmed with pre-cancer diseases. Follow-up is no-going with remaining individuals. CDA test results on Caucasian group showed comparable sensitivity and specificity as those on Chinese Han ethnic group.
TABLE-US-00013 TABLE 12A Cancer Cases Number Percent Lung cancer 14 19% Colorectal cancer 14 19% Prostate cancer 9 12% Gastric cancer 6 8% Breast cancer 5 7% Esophageal cancer 3 4% Lymphoma cancer 3 4% Cutaneum carcinoma 3 4% Renal carcinoma 3 4% Liver cancer 2 4% Pancreatic cancer 2 3% Cancerous goiter 2 3% Cervical cancer 2 3% Bladder cancer 1 1% Tonsillar Carcinoma 1 1% Osteocarcinoma 1 1% Leukaemia 1 1%
TABLE-US-00014 TABLE 12B Non-Cancerous Disease Number percent Pulmonary nodule 27 24% Gastroduodenal diseases 25 22% Thyroid nodule 17 15% Hysteromyoma 11 10% Liver disease 8 7% Colorectal polyp 7 6% Renal cyst 5 4% Breast disease 5 4% Prostatic cyst 2 2% Gallbladder polyps 2 2% Oophoritic cyst 2 2% Enteric adenoma 1 1% Meningioma 1 1%
[0364]
[0365] While the functions and properties of bio-physics have played a critical role in physiology, they have not been extensively utilized in the field of IVD of cancer, which has traditionally been more heavily replied upon bio-chemistry, immunology, and genomics. This work represents a novel approach and breakthrough in the field cancer detection. Results demonstrated that this technology has unique advantage to detect cancer early, and can be an effective approach to track disease progression, as it showed statistical difference between healthy group and disease group, and between disease group and cancer group. Compared with traditional approaches, the current approach detects a signal which is much more foundational and it is in existence in all human being including healthy individuals. Therefore, its signal is much earlier in nature in detecting occurrence of cancer. Further, micro-electrical current has shown to decrease significantly from healthy group to disease group and from disease to cancer group, making it ideal for early stage cancer detection and tracking diseases leading to cancer.
[0366] Results from tests (a) using samples with increasing amount of cancer cells, (b) using samples with increasing amount of bio-marker concentration CEA, and (c) with samples with and without an assay which is known to cause a molecular level reaction showed that CDA values are proportional to increasing amount of cancer cells and bio-marker CEA concentrations. In addition, CDA values are dependent on with and without molecular level reactions. Based on the above observations, it can be stated that CDA values are a function of cellular, protein, and molecular levels (as shown in
[0367]
[0368]
[0369]
[0370] Having demonstrated viability of this new technology for pre-cancer and early stage cancer detection, possible mechanism can be further proposed. A scheme of cells, proteins, and genetic components (DNA, RNA, etc.) and their surrounding liquid media (e.g., blood) is described above and provided in
[0371] Compared with other traditional cancer detection technologies, CDA technology has many unique features and clear advantages. First, many existing technologies detect cancer signals after cancer has already formed which make those technologies ineffective for early stage cancer detection, while CDA technology detects a bio-physical parameter which exists in healthy individuals and rises as the risk of cancer increases (as shown in
[0372] In addition, based on CDA value dependent disease progression behavior (disease progresses with decreasing micro-electrical current of the blood sample); based on the above proposed hypothesis, new model for cancer occurrence is proposed as follows. In this new model, as a major bio-physical parameter, the shift in electrical properties of blood, specifically, decreasing in micro-electrical current and/or changing quantum mechanical effects (which affect gene replications and mutations) is causing negative effects at multi-levels which include (1) reduced surface charge, cell repulsion, and cell signaling efficiency at cellular level, and (2) reduced electrostatic force, DNA surface charge, and possibly increased mutation at DNA level. Further, it is hypothesized that reduced micro-electrical current (and conductance) also causes reduced surveillance capability of T cells for cancer cell detection and reduced immunity which increase occurrence of cancer. The above hypothesis is supported by data collected in this work showing that decreasing (increasing CDA values) in micro-electrical current is correlated with disease progress from healthy group to disease group, from disease group to pre-cancer group, and from pre-cancer group to cancer group.
[0373]
[0374] In this invention, changes in electrical properties in blood and DNA level can be used as a tool for disease detection. As electrical current and conductance decrease, a number of molecular level (DNA surface charge decreases, quantum mechanical effect change, and DNA mutation increases) properties degrade, resulting in increased disease and cancer occurrence. As shown in
[0375] Furthermore, the new technology according to this invention can also be used in assisting in diagnosis, such as assisting in diagnosis of lung cancer. As shown in
[0376] As also shown in
[0377] Initial clinical study results show that the novel technology according to this invention is capable of evaluating effectiveness of drug treatment of cancer. In this case (e.g., as shown in
[0378] One of key aspects of this invention is that the bio-physical properties and its associated behaviors disclosed in this novel work are of common to a large number of cancer types, and can be used for detection of a large number of cancer types, making the disclosed method a viable technology for cancer screening, assisting in diagnosis, prognosis, therapy selection and reoccurrence detection.
[0379]
[0380]
[0381] As shown in
[0382] In one embodiment, utilizing a micro-fluidic device with micro-channels and sensitive sensors, electrical properties of blood samples at near field of cells illustrated in above figure (schematic of cellular membranes) can be measured, and related electrical properties including electrical current across the region, trans-membrane potential, and ion levels (potassium ions, sodium ions, chloride ions, calcium ions, and nitride ions) can be directly and indirectly measured. Since disease state of mammals is related to the above-mentioned cellular bio-physical properties (and DNA, RNA and other biological entities in the cells), the above inventive measurement technology can be used to detect diseases including pre-cancer and cancer diseases. The membrane potential can regulate the balance between normal cellular activities including normal growth and replications, and carcinogenesis. As such, both ion level and concentration (potassium ions, sodium ions, chloride ions and calcium ions) and membrane potential could be used as a new, novel bio-marker for cancer prevention and early stage cancer detection.
[0383] The present invention provides a new cancer detection technology using a bio-physical approach based on electrical properties of liquid samples for IVD applications. In this new technology, a micro-electrical current is detected which has shown to be very effective in detecting pre-cancer and early stage cancer. This technology has the advantages of detecting cancer early, high sensitivity and specificity, covering a wide range of cancer types, and relatively simple and cost effective. Based on how CDA values are correlated to control, disease and cancer groups in this work, and possible effects of electrical properties in blood on disease progression, a new hypothesis on cancer occurrence model is proposed in which a reduction in blood micro electrical current (and conductance) and/or a change of quantum mechanical effects is proposed to cause a number of negative effects at cellular and molecular levels, resulting in reduced cell to cell signaling, cell to cell repulsion, and immunity, and increased gene mutation frequency, and hence increased occurrence of cancer.
[0384] While for the purposes of demonstration and illustration, the above cited novel, detailed examples show how microelectronics and/or nano-fabrication techniques and associated process flows can be utilized to fabricate highly sensitive, multi-functional, powerful, and miniaturized detection devices, the principle and general approaches of employing microelectronics and nano-fabrication technologies in the design and fabrication of high performance detection devices have been contemplated and taught, which can and should be expanded to various combination of fabrication processes including but not limited to thin film deposition, patterning (lithography and etch), planarization (including chemical mechanical polishing), ion implantation, diffusion, cleaning, various materials, combination of processes and steps, and various process sequences and flows. For example, in alternative detection device design and fabrication process flows, the number of materials involved can be fewer than or exceed four materials (which have been utilized in the above example), and the number of process steps can be fewer or more than those demonstrated process sequences, depending on specific needs and performance targets. For example, in some disease detection applications, a fifth material such as a biomaterial-based thin film can be used to coat a metal detection tip to enhance contact between the detection tip and a biological subject being measured, thereby improving measurement sensitivity.
[0385] Applications for the detection apparatus and methods of this invention include detection of diseases (e.g., in their early stage), particularly for serious diseases like cancer. Since cancer cell and normal cell differ in a number of ways including differences in possible microscopic properties such as electrical potential, surface charge, density, adhesion, and pH, novel micro-devices disclosed herein are capable of detecting these differences and therefore applicable for enhanced capability to detect diseases (e.g., for cancer), particularly in their early stage. In addition to micro-devices for measuring electrical potential and electrical charge parameters, micro-devices capable of carrying out mechanical property measurements (e.g., density) can also be fabricated and used as disclosed herein. In mechanical property measurement for early stage disease detection, the focus will be on the mechanical properties that likely differentiate disease or cancerous cells from normal cell. As an example, one can differentiate cancerous cells from normal cells by using a detection apparatus of this invention that is integrated with micro-devices capable of carrying out micro-indentation measurements.
[0386] Although specific embodiments of this invention have been illustrated herein, it will be appreciated by those skilled in the art that any modifications and variations can be made without departing from the spirit of the invention. The examples and illustrations above are not intended to limit the scope of this invention. Any combination of detection apparatus, micro-devices, fabrication processes, and applications of this invention, along with any obvious their extension or analogs, are within the scope of this invention. Further, it is intended that this invention encompass any arrangement, which is calculated to achieve that same purpose, and all such variations and modifications as fall within the scope of the appended claims.
[0387] All publications or patent applications referred to above are incorporated herein by reference in their entireties. All the features disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example of a generic series of equivalent or similar features.