JP2022007854A - System and method for automatic detection of cloud base detecting causal organism and antibody array by point of care (poc) - Google Patents

Abstract

To provide an automatic detection system and a method for a cloud base for detecting a causal organism and an antibody array by point of care (POC).SOLUTION: An illustrated embodiment of an invention includes an automatic method for assaying virus and antibody specimen in a sample in a portable hand held micro fluid reader having an SAW detector having a minimum mass sensitivity limit. The automatic method includes a step for automatically executing assay by using an SAW detector with enhanced sensitivity like Optikus I but also including: a step for automatically arranging a second portion of the sample on a micro array; a step for automatically and selectively probing a second portion of the sample regarding an antibody corresponding to at least one selected virus by using the micro array; and a step for discriminating an antibody in the second portion of the sample by automatically reading the micro array by using a fluorescent camera.SELECTED DRAWING: None

Description

[01] This application is U.S. Patent Application No. 16/714 entitled APPARATUS AND METHOD FOR OVERCOMING MINIMAL MASS SENSITIVITY LIMITATIONS IN A SHEAR HORIZONTAL SURFACE ACOUSTIC WAVE BIOSENSOR filed on December 13, 2019 in accordance with Article 120 of the U.S. Patent Act. , 421 is a partial continuation application, claiming its priority and the benefits of retroactive filing date, and all content of this US patent application is incorporated herein by reference ("Incorporated". Defined as "specification").

[02] Background
[03] Field of technology
[04] The present invention relates to the field of point of care (POC) pathogen and multiplexed pathogen antibody array detection platforms and methods, particularly CPC C40B 60/12.

[05] Explanation of prior art
[06] Over the past few decades, an increasing number of emerging infectious diseases have caused serious social and economic consequences on a global scale. In particular, rural Third World communities are not only experiencing high exposure to infectious diseases, but also face many challenges in medical access. Nevertheless, pathogens do not identify borders, and outbreaks of new diseases affect people everywhere, wherever they are. Expertly selected knowledge, software, and services that help humanity interpret medical diagnostic test results from interconnected Point of Care networks around the world to track and prevent the rapid spread of infectious disease pandemics. It is the only way we can expect to maintain a vibrant economy and a highly fluid society.

[07] What is needed is a device that addresses some of the most pressing requirements of establishing disease screening, interpretation, and prevention goals by using the current COVID-19 pandemic as the most pressing target. And the method. Coronavirus is an enveloped plus-strand RNA virus that is widely distributed in humans, other mammals, and birds and causes respiratory, intestinal, liver, and neurological disorders. There are six known species of this virus that are known to infect humans, but only four are endemic and usually cause cold-like symptoms: 229E, OC43, NL63, and HKU1. The other two viruses, the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV), are common human and animal infectious viruses, 2002-2003 and 2012. It caused a big pandemic event in the year. The global coronavirus pandemic, the first case of which was detected and reported at the end of 2019, is a novel, named SARS-CoV-2 by the World Health Organization (WHO), which causes COVID-19 disease. It is the result of the coronavirus. Viral surface proteins (spikes, envelopes, and membranes) are embedded in the host cell-derived lipid bilayer envelope. Single-strand plus-strand viral RNA is associated with nucleocapsid proteins as shown in FIG.

[08] Current POC COVID-19 detection platforms are divided into four categories: 1) SARS-CoV-2 detection methods, 2) ELISA, immunofluorescence assay, 3) RT-qPCR, and 4) chest X-ray. .. Chest x-rays are not practical for popular POC examinations.

[09] Enzyme-linked immunosorbent assay-ELISA.
[10] Serological tests measure antibodies in the blood from people exposed to the virus. ELISA blood tests check for immunoglobulins (IgG) resulting from past or recent exposure to coronavirus disease 2019 (COVID-19). The human body produces IgG antibodies as part of an immune response to the virus. It usually takes about 10 to about 18 days to produce sufficient antibodies for detection in blood. In addition, ELISA blood tests can look for immunoglobulin M (IgM) antibodies, which are the first antibodies to appear after an individual is exposed to an antigen, but disappear when the antigen is absent. ELISA, which looks at IgG and IgM at the same time, can represent the reality of the disease the patient is currently fighting for and the disease the patient already has and has acquired immunity.

[11] The commonly used sample is a blood sample that is generally collected more reliably than a nasal swab or pharyngeal swab sample (used for test types 2 and 3 above). However, for blood samples, handling, storage, and centrifugation to separate serum from plasma are necessary additional steps that can introduce errors. If the antibody is found in a serological test sample, a direct immunoassay (see test type 2) or DNA test (see test type 3) is performed to establish whether the virus itself is still present in the patient's body. ..

[12]
[13] Immunofluorescence assay.
[14] These assays have been widely used to directly detect a wide variety of viral antigens. Immunofluorescence uses antibodies to detect viral antigens in tissue sections or infected cells. Infected cells from the mucosa of the upper respiratory tract or cells present in the mucus aspirated from the nasopharynx are used and collected using a cotton swab. The immunofluorescence assay uses a fluorescent label coupled to an anti-viral antibody, which is known as direct immunofluorescence, or a fluorescent label coupled to an anti-antibody, which is known as indirect immunofluorescence. .. The amount of antibody bound to the antigen directly correlates with the amount of fluorescence source.

[15] We have introduced an alternative direct, unlabeled virus detection technique based on SAW sensors. See the referenced specification. The sensor utilizes a surface acoustic wave biosensor (SAW) for direct COVID-19 detection. Traditionally, SKC SAW sensors have been successful in detecting many high profile bacteria and viruses, including Ebola, HIV, and anthrax. Over the last two years, the sensitivity and detection capabilities of SAW biosensors have been significantly improved. The SARS-CoV-2 antibody is immobilized on the SAW surface and the reaction as a function of concentration is evaluated. A high-speed (<12 minutes) point-of-care diagnostic test that detects COVID-19 from a swab sample for the nose.

[16] The use of cotton swabs is not necessarily due to a low sensitivity detection methodology, and the inability to reproducibly collect samples from the nasal meatus results in a significant number of false negatives. There are variations among the medical staff who collect the samples, and the amount of virus present in the nose also varies. Another major drawback is that these tests give only positive results if the virus is still present. The test cannot identify people who have recovered from the infection and are free of the virus from their bodies.

[17]
[18] Real-time quantitative polymerase chain reaction-RT-qPCR
[19] Direct pathogen detection based on fast DNA amplification. PCR is used to measure the quantity of genetic material (DNA or RNA) in a sample, involving the use of Taq polymerase, which amplifies a short specific portion of template DNA in a temperature cycle. At each cycle, some small specific sections of DNA are doubled, resulting in exponential amplification of the target. The number of cycles in a PCR experiment is usually 12-45 cycles. Since RNA is the reverse transcription of DNA, reverse transcriptase PCR (RT-PCR) is used to detect RNA. In RT-qPCR, the same method is performed except for two factors: i) the amplified DNA is fluorescently labeled and ii) the amount of fluorescence released during amplification is directly linked to the amount of amplified DNA source. Will be done. The one-step RT-qPCR detection kit is useful for in vitro detection of COVID-19 using a respiratory specimen-nasal swab. An example of a POC product is the ID NOW branded Abbott coronavirus test using testing equipment.

[20] What is needed is a technique that solves the problems associated with currently available COVID-19 diagnostic or testing equipment.

overview
[21] The illustrated embodiment of the present invention relates to a cloud-based automated system using a handheld field-portable diagnostic device capable of automatically performing laboratory-grade diagnostic tests for viral pandemic infections. After taking a biological sample and placing it in a handheld field portable diagnostic instrument, all further steps of sample preparation and testing are performed automatically without human intervention and associated delays. Within tens of minutes from the test, without the need or delay of human intervention or further medical intervention, the sample is tested, the results are generated, analyzed by artificial intelligence or a professional system, communicated to the memory database and tested. It is communicated to the subject and to the relevant health care provider. Only in this form is it possible to provide reliable pandemic testing and reporting of hundreds of millions of subjects, which is a necessary capability if a global pandemic should be stopped or controlled.

[22] The illustrated embodiment of the invention comprises assaying a virus and antibody sample in a sample in a portable handheld microfluidic reader with a detector having a minimum mass sensitivity limit. The method captures a sample from a step of inserting the sample into a reader and a first portion of the sample having a first antibody with attached DNA tags and a second antibody with attached magnetic nanoparticles (MNPs). By providing a capture step in which a sandwich containing the first and second antibodies, specimens, MNPs, and DNA tags is formed, and an amount reliably detectable by the detector. Amplification is used to replicate the DNA tag to a predetermined amount of DNA tag sufficient to overcome the detector's mass sensitivity limit, and the detector is used to measure the amount of replicated DNA tag. , A step of detecting at least one selected virus, a step of placing a second portion of the sample on a microarray, and a second part of the sample for antibodies corresponding to at least one selected virus using the microarray. It comprises the step of selectively probing the portion of the sample and the step of reading the microarray using a fluorescent camera to identify the antibody in the second portion of the sample.

[23] The step of selectively probing a second portion of a sample for an antibody corresponding to at least one selected virus using a microarray is a second step of the sample on the microarray over a predetermined amount of time. It includes a step of culturing a portion, a step of placing a fluorescently labeled secondary Ab, a step of washing the microarray, and a step of drying the microarray. The step of reading the microarray using a fluorescent camera and identifying the antibody in the second part of the sample detects the Ig isotype in the second part of the sample by generating a color image of the microarray. The method further comprises communicating the color image of the microarray to the cloud for analysis and / or data processing.

[24] In embodiments where the microarray is provided with DNA spots for the receptacle binding domain (RBD) of the spike protein, the microarray is used to use a second sample of antibody corresponding to at least one selected virus. The step of selectively probing moieties is a microarray provided with DNA spots for the receptacle binding domain (RBD) of the spiked protein, fluorescently labeled ACE2, and another fluorescently labeled to human IgG to detect RBD antibody. It includes a step of performing a neutralization antibody assay using the secondary antibody, a step of washing the microarray, and a step of drying the microarray. The step of reading the microarray using a fluorescent camera and identifying the antibody in the second part of the sample produces a color image of the microarray with at least two different colors, one color present in the second part of the sample. For RBG antibody, the second color is for ACE2, and the second portion of the sample without neutralizing antibody or RBD antibody is detected using ACE2 fluorescence, while the sample with RBD antibody or ACE2. -Samples with a large amount of neutralizing antibody that interferes with RBD binding are detected by reducing the amount of ACE2 fluorescence. In the absence of RBD antibody, the amount of ACE2 fluorescence can be quantified for relative neutralizing activity. The method further comprises communicating the color image of the microarray to the cloud for analysis and / or data processing.

[25] Microarrays include normalized fluorescence controls that fluoresce at known intensities. Three spots are 100% strong, three spots are 50% strong, and three spots are 0% strong. By measuring these nine spots, it is possible to create a normalized curve that can be matched when comparing and plotting the remaining inspection dots.

[26] The step of selectively probing a second portion of a sample for an antibody corresponding to at least one selected virus using a microarray is the step of micromixing the second portion of the sample using reciprocating motion. The centrifugal acceleration acting on the liquid element first produces and stores air energy, which is then released by the decrease in centrifugal acceleration, resulting in the opposite direction of liquid flow. A sequence of alternating high and low centrifugal accelerations is applied to the second portion of the sample to culture / culture between antibody macromolecules and antigen macromolecules during hybridization. It further comprises a step of maximizing hybridization efficiency.

[27] The illustrated embodiment also includes a portable handheld microfluidic reader assaying a sample in a sample. The reader has a rotatable microfluidic disk or rotor with a sample inlet defined on the disk into which the sample is inserted and a sample defined on the disk and selectively communicated with the sample inlet to which a DNA tag is attached. A mixing chamber provided with a first antibody to capture and a mixing chamber provided with a second antibody defined on a disk and having a sample attached to the surface or having magnetic nanoparticles (MNP) attached. An amplification chamber that selectively communicates, a sandwich containing a surface or MNP, first and second antibodies, specimens and DNA tags is formed in the amplification chamber and replicates the DNA tags using isothermal amplification. A second portion of the sample, an amplification chamber that produces a predetermined amount of DNA tag, and a detector provided on the disk to selectively communicate with the amplification chamber and measure the amount of replicated DNA tag. A receiving reaction chamber, a microarray placed in the reaction chamber and selectively probing a second portion of the sample for an antibody corresponding to at least one selected virus using the microarray, and a microarray reading the microarray of the sample. Includes a fluorescent camera to identify the antibody in the second part.

[28] The detector includes an LED that excites and measures the fluorophore in the microarray on disk CD-1, and includes a CMOS camera that captures photons emitted by the excited fluorofore. The device also includes two filters with separate wavelength bands, one between the fluorofore and the microarray (750 nm) and the other between the microarray and the CMOS camera (790 nm). The bands of the two filters are separated and do not share any overlap in the electromagnetic spectrum, thus ensuring that any scattered light from the emitting LED does not affect the measurements made by the CMOS camera.

[29] The detector includes a surface acoustic wave (SAW) detector used in combination with CD-2. The detector includes all necessary RF signal generators and interposers to interface with the device and disk, thereby allowing RF measurements to be made on the SAW sensor.

[30] Disc CD-3 performs the LAMP isothermal PCR detection methodology disclosed in the incorporated specification, and the fluid sample changes color based on the amount of DNA in the initial sample. In the illustrated embodiment, the disc does not have more than one type of detector, but the scope of the invention extends to the possibility of providing a disc with a combination of different types of detectors. ..

[31] In an embodiment where the sample is a blood sample, the reader is the plasma containing the sample at the inlet communicating with the sample inlet and from the first part of the sample to the mixing chamber and from the second part of the sample to the reaction chamber. Further includes a plasma-blood separation chamber with an outlet to transmit.

[32] The detector includes a barcode reader that scans the barcode printed on the disposable disc package to collect information about the particular type of inspection being performed. In addition, the barcode reader can read the barcode on the smart device screen or patient wristband to identify and log the results corresponding to a particular patient.

[33] The detector is a TCP / IP Wi-Fi module that wirelessly transmits the information collected by the detector to the cloud infrastructure and receives the information transmitted by the cloud infrastructure such as patient information, result analysis, and software updates. including.

[34] The detector includes a Bluetooth module that performs both data transmission and wireless transmission of the results and remote control of the detector's wireless operation.

[35] The detector includes a capacitive touch screen that interacts with the detector and displays the test results to the user. The detector screen includes a graphical user interface, which includes the steps necessary to guide the user through the process of collecting the sample, inserting the microfluid into the device, and displaying the final result of the test.

[36] The detector includes a Pelche temperature control element that interfaces with the microfluidic disk to maintain thermal equilibrium.

[37] To recapitulate, the illustrated embodiment of the invention is a method of field diagnostic testing of a sample taken from a subject in a portable handheld device to determine the presence of viral antigens and / or antibodies. Using the instrument according to the nature of the sample and the corresponding detection means within the instrument of the viral antigen and / or antibody to be diagnostically tested, the step of placing the sample in the receiving chamber of the rotatable disk of the rotatable disk. In, the step of selectively processing the sample, the step of detecting the quantitative measurement of the viral antigen and / or the antibody in the sample using the corresponding detection means in the instrument, and the step of detecting the viral antigen in the sample corresponding to the subject. And / or the step of generating the data output of the detected quantitative measurement of the antibody, the step of communicating the data output corresponding to the subject to the cloud-based database, and several different types of viral antigens and / or in the cloud-based ecosystem. / Or a step of comparing and analyzing the antibody and the communicated data output corresponding to the subject to diagnose the type of viral infection, if any, has the highest or previous viral infection that the subject has. And include methods of communicating the results of the comparative analysis from the cloud-based ecosystem to the subject.

[38] Detection means include microarrays of antigen and / or antibody fluorescent spots. The step of detecting the quantitative measurement of the viral antigen and / or antibody in the sample using the corresponding detection means in the instrument is the step of generating an image file of a color image of a microarray of antigen and / or antibody fluorescent spots. include.

[39] In another embodiment, the detection means includes a functionalized surface acoustic wave detector (SAW). The step of detecting the quantitative measurement of the viral antigen and / or antibody in the sample using the corresponding detection means in the instrument is directly captured by the functionalized surface elastic wave detector (SAW) or the viral antigen and / or. Alternatively, an RF phase-delayed detection signal in response to quantification of viral antigens and / or antibodies indirectly captured by a polymerase chain reaction (PCR) replicating DNA tag by a functionalized surface elastic wave detector (SAW) corresponding to the antibody. Includes generating.

[40] The step of selectively processing a sample on a rotatable disc using the instrument according to the nature of the sample and using the corresponding detection means within the instrument of the viral antigen and / or antibody to be diagnostically tested is immunoimmuno. It comprises performing an ELISA blood test check for globulin G (IgG) antibody and immunoglobulin M (IgM) antibody.

[41] In another embodiment, the instrument is used according to the nature of the sample and the sample is selectively selected on a rotatable disc using the corresponding detection means within the instrument for the viral antigen and / or antibody to be diagnostically tested. The processing step comprises performing an immunofluorescence assay using conjugated fluorescence labeling by direct or indirect immunofluorescence, in which the amount of antibody conjugated to the antigen directly correlates with the amount of fluorescence source.

[42] In yet another embodiment, the instrument is used according to the nature of the sample and the sample is selectively selected on a rotatable disk using the corresponding detection means within the instrument of the viral antigen and / or antibody to be diagnostically tested. The step to process is to measure the amount of genetic material (DNA or RNA) in the sample using PCR and to perform a real-time quantitative polymerase chain reaction (RT-qPCR) by fast DNA amplification using Taq polymerase. Includes steps to perform.

[43] The step of selectively processing a sample on a rotatable disk using the instrument according to the nature of the sample and using the corresponding detection means within the instrument for viral antigens and / or antibodies to be diagnostically tested is the sample. A step of capturing a sample having a first antibody to which a DNA tag is attached and a second antibody having magnetic nanoparticles (MNP) attached to the DNA tag from the first portion of the first antibody and the second. Using isothermal amplification, the DNA tag of the detector is provided with a capture step in which a sandwich containing the antibody, sample, MNP, and DNA tag is formed, and an amount that is reliably detectable by the detector. The step of replicating to a predetermined amount of DNA tag sufficient to overcome the mass sensitivity limit, the step of placing a second portion of the sample on a microarray, and the use of a microarray to accommodate at least one selected virus. It comprises the steps of selectively probing a second portion of the sample for the antibody and reading the microarray using a fluorescent camera to identify the antibody in the second portion of the sample.

[44] In yet another embodiment, the step of selectively probing a second portion of a sample for an antibody corresponding to at least one selected virus using a microarray is on the microarray for a predetermined amount of time. Includes a step of culturing a second portion of the sample, placing a fluorescently labeled secondary Ab, washing the microarray, and drying the microarray. The steps of reading the microarray using a fluorescent camera and identifying the antibody in the second part of the sample include the step of detecting the Ig isotype in the second part of the sample by generating a color image of the microarray. Includes the steps of communicating a color image of a microarray to the cloud for analysis and / or data processing.

[45] In one embodiment, the microarray is provided with a DNA spot for the receptacle binding domain (RBD) of the spike protein, and the microarray is used to sample the antibody corresponding to at least one selected virus. The step of selectively probing the second portion is a microarray provided with DNA spots for the receptacle binding domain (RBD) of the spike protein, fluorescently labeled ACE2, and another fluorescence against human IgG to detect the RBD antibody. It comprises performing a neutralizing antibody assay using the labeled secondary antibody, washing the microarray, and drying the microarray. The step of reading the microarray using a fluorescent camera and identifying the antibody in the second part of the sample is the step of generating a color image of the microarray having at least two different colors, one color being the second part of the sample. The second part of the sample without neutralizing antibody or RBD antibody is detected using ACE2 fluorescence, while the RBD antibody is for the RBG antibody present in the portion of, the second color is for ACE2. A sample having or a sample having a large amount of neutralizing antibody that interferes with ACE2-RBD binding is detected by reducing the amount of ACE2 fluorescence, and in the absence of RBD antibody, the amount of ACE2 fluorescence should be quantified for relative neutralization activity. Includes the steps to generate and communicate the color image of the microarray to the cloud for analysis and / or data processing.

[46] The step of selectively probing a second portion of a sample for an antibody corresponding to at least one selected virus using a microarray is to micromix the second portion of the sample using reciprocating motion. The centrifugal acceleration acting on the liquid element first produces and stores air energy, which is then released by the decrease in centrifugal acceleration, resulting in the opposite direction of liquid flow. A sequence of alternating high and low centrifugal accelerations is applied to the second portion of the sample to culture / culture between antibody macromolecules and antigen macromolecules during hybridization. It further comprises a step of maximizing hybridization efficiency.

[47] In a cloud-based ecosystem, comparing and analyzing multiple different types of viral antigens and / or antibodies and the communicated data output corresponding to the subject, the subject has the highest or previous probability of having it. If there is the highest viral infection, the steps to diagnose the type of viral infection are to analyze the microarray's communicated data output for positive and / or negative indications for the COVID-19 antigen and / or antibody, and to COVID-. Data output communicated for 19 positive and / or negative instructions with data output communicated for positive and / or negative instructions of multiple viral infection microarrays that share at least some of the COVID-19 antigen and / or antibody. Does the compared step and the communicated data output of the positive and / or negative indication statistically indicate COVID-19 rather than multiple viral infections that share at least some of the COVID-19 antigen and / or antibody? It involves determining whether or not, thereby significantly reducing false positives and / or false negatives.

[48] Whether the communicated data output of positive and / or negative indications statistically indicates COVID-19 rather than multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. The step of determining is that the corresponding Z-score of the communicated data output of the positive and / or negative indication is COVID rather than the Z-score of multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. Includes a step to determine whether to indicate -19.

[49] Communicate the data output communicated for positive and / or negative indications for COVID-19 for positive and / or negative indications for microarrays of multiple viral infections that share at least some of the COVID-19 antigens and / or antibodies. The step of comparing the data output to the SARS-CoV-2, SARS-CoV, MERS-CoV, common cold coronavirus (HKU1) is to compare the data output communicated for the positive and / or negative indication of COVID-19. , OC43, NL63, 229E), and a microarray of multiple acute respiratory infections selected from the group comprising multiple subtypes of influenza, adenovirus, metapneumovirus, parainfluenza, and / or respiratory conjugate virus. Includes a step of comparing the positive and / or negative indications of the virus to the communicated data output.

[50] Whether the communicated data output of positive and / or negative indications statistically indicates COVID-19 rather than multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. The step of determining, which significantly reduces false positives and / or false negatives, uses the recipient motion characteristic (ROC) curve from which the sub-curve area (AUC) is measured. The steps include assessing the antigen, distinguishing output data from the antigen-negative group to the antigen-positive group over a wide range of assay cutoff values, and identifying high-performance antigens that diagnose COVID-19.

[51] Whether the communicated data output of positive and / or negative indications statistically indicates COVID-19 rather than multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. The step of determining, thereby significantly reducing false positives and / or false negatives, is a plurality of determination steps based on the corresponding Ioden index calculated with a combination of multiple high performance antigens. Includes steps to identify optimal sensitivity and specificity for COVID-19 from a combination of high performance antigens.

[52] The scope of the illustrated embodiments of the invention is also extended to portable handheld microfluidic readers that assay specimens in a sample, the reader having a rotatable microfluidic disc. The reader is a mixing chamber provided with a sample inlet defined on the disk on which the sample is placed and a first antibody defined on the disk and selectively communicated with the sample inlet to capture the sample to which the DNA tag is attached. An amplification chamber that is defined on a disk and selectively communicates with a mixing chamber provided with a second antibody that captures a sample that adheres to the surface or has magnetic nanoparticles (MNPs) that adhere to the surface. Alternatively, with an amplification chamber, a sandwich containing MNP, first and second antibodies, specimens and DNA tags is formed in the amplification chamber and replicates the DNA tags using isothermal amplification to produce a predetermined amount of DNA tags. , A detector provided on the disk to selectively communicate with the amplification chamber and measure the amount of replicated DNA tag, a reaction chamber to receive a second portion of the sample, and a microarray located in the reaction chamber. A microarray that selectively probates a second portion of the sample for antibodies corresponding to at least one selected virus, and a fluorescent camera that reads the microarray and identifies the antibody in the second portion of the sample. including.

[53] In one embodiment, the detector is a surface acoustic wave (SAW) detector.

[54] In one embodiment, the sample is a blood sample and the reader is the inlet communicating with the sample inlet and the sample from the first part of the sample into the mixing chamber and from the second part of the sample into the reaction chamber. Includes a plasma-blood separation chamber with an outlet for transmitting plasma containing.

[55] The illustrated embodiment can also be assayed for a sample in a sample and characterized as a portable handheld microfluidic reader operating in a cloud ecosystem. The reader has a rotatable microfluidic disk, and the reader has a fluid engineering circuit in the disk on which the sample is placed and processed, multiple fluorescently tagged antigens and / or antigen probes placed in the fluid engineering circuit. Microarrays that selectively probe samples for antibodies and / or antigens corresponding to at least one selected virus, fluorescent color cameras that image the microarrays to identify antibodies and / or antigens in the samples, and microarrays. It includes a circuit that generates output data corresponding to an image, quantifies the amount of antigen and / or antibody probed and detected in a sample, and communicates the output data to a cloud ecosystem.

[56] The portable handheld microfluidic reader receives output data from cloud-based databases and readers and provides evidence of existing or past viral infections from the probed and detected antigens and / or antibodies in the sample. Further includes a combination of processors for statistical diagnosis.

[57] The device and the method will be described or described for grammatical fluency along with a functional description, but the claims are expressly under Section 112 of the United States Patent Act. Unless formulated, it should never be construed as inevitably limited by the construction of "means" or "step" limitations, and is provided by the claims under the doctrine of equality. The full scope and equivalent of the provisions of the above provisions should be followed, and if the claims are expressly defined under Article 112 of the US Patent Act, the maximum statutory provisions under Article 112 of the US Patent Act. It should be explicitly understood that the equality should be obeyed. The present disclosure can now be better visualized by reference to the following drawings in which similar elements are referenced by similar numbers.

A brief description of the drawing
[58] The patent or application file contains at least one color drawing. A copy of this patent or patent application gazette with color drawings will be provided by the Patent Office upon request and payment.

[59] A graph showing IgG serum reactivity as measured by the fluorescence intensity of serum specimens on a coronavirus antigen microarray relative to frequency, the array being performed on disk CD-1 on the Optikus platform, FIG. 1a. Fluorescence spectrophotographic segments corresponding to multiple viruses shown side by side. [59] A graph showing IgG serum reactivity as measured by the fluorescence intensity of serum specimens on a coronavirus antigen microarray relative to frequency, the array being performed on disk CD-1 on the Optikus platform, FIG. 1b. Expansion of the spectrum of COVID-19 protein, SARS-CoV protein, and MERS-COVID protein. [60] A heatmap of coronavirus antigen microarrays, which are organized into columns classified as positive or red (recovery from PCR-positive individuals) or negative or blue (from uninfected individuals before pandemics). The serum showed IgG reactivity in FIG. 2a and IgA reactivity in FIG. 2b measured as average fluorescence intensity over four replications matched against each DNA dot in a microarray organized in virus-colored rows, showing reactivity. Is presented in color below the heatmap (white = low, black = medium, red = high). [60] A heatmap of coronavirus antigen microarrays, which are organized into columns classified as positive or red (recovery from PCR-positive individuals) or negative or blue (from uninfected individuals before pandemics). The serum showed IgG reactivity in FIG. 2a and IgA reactivity in FIG. 2b measured as average fluorescence intensity over four replications matched against each DNA dot in a microarray organized in virus-colored rows, showing reactivity. Is presented in color below the heatmap (white = low, black = medium, red = high). [61] Graphs of normalized IgG reactivity of positive and negative sera on coronavirus antigen microarrays, plots from convalescent sera (positive, red) from PCR-positive individuals and uninfected individuals before pandemics. The heat map below the plot shows the IgG reactivity for each antigen measured as mean fluorescence intensity (MFI) with full range (bar) and quadrant (box) in serum (negative, blue). The average reactivity of each group is shown (white = low, black = medium, red = high), and the antigen labeling is colored in each virus group. [62] A perspective partial transparent view of Optikus II that processes viral samples to quantify the antigen-specific antibody response induced after infection, and the sample preparation step-depending on the assay-is sample introduction, blood plasma. Includes isolation, plasma isolation, amplification or culture, washing, and measurement (SAW detection for viruses and fluorescence intensity detection for antibodies). [63] FIG. 6 illustrates an assay procedure on a microarray performed on a laboratory benchtop. [64] It is a figure which shows the method of the antiviral Ab detection in a blood using an antigen array, or the virus antigen detection in a nasal discharge by an antibody array. [65] FIG. 6 is a diagram of a method of using an antibody neutralizing assay using an array of RBD antigens. [66] It is a schematic diagram of the fluid engineering system in the disk CD-1. [66] Schematic representation of antibody capture at each array element, the reaction chamber holds a spotted protein array. [67] In a graph of experimental results comparing the strength of an IgG microarray made using a reciprocating flow system with different culture times with the strength of an IgG microarray made using a manual method with a culture time of 1 hour. be. [68] Compare the characteristics of reciprocating flow in the illustrated embodiments of culture compared to single flow and passive diffusion. [68] Compare the characteristics of reciprocating flow in the illustrated embodiments of culture compared to single flow and passive diffusion. [68] Compare the characteristics of reciprocating flow in the illustrated embodiments of culture compared to single flow and passive diffusion. [69] FIG. 6 is a diagram of a fluorescent biosensor of an illustrated embodiment of a protein array having a CMOS laser / diode chip underneath for signal readout on disk CD-1. [70] FIG. 6 shows a microlancet and a 300 μL microbette into which a blood sample has been drawn. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [72] FIG. 6 is a diagram of a CD rotor containing two SAW detectors corresponding to the fluid engineering circuit and RF interposer on disc CD-2. [73] This is a diagram of the Optician II cloud platform. [74] It is a figure which shows the usage of Optician II using a cloud infrastructure. [75] It is a diagram of the structure of a novel coronavirus. [76] [77] Graphs of individual patient fluorescence scores for IgG and IgM detection in samples using microarrays and the corresponding Z-score statistics, the red line is an assessment of whether the measurement is positive or not. The blue line is the average of the negative results, the red corresponds to the average serologic positive result further confirmed via PCR, and the blue line is confirmed via PCR. If the patient's IgG bar graph looks like a red line, the test is positive, if it looks like a blue line, the test is negative, and FIG. 18a is shown in FIG. 18c. Several viruses, namely SARS-CoV-2, SARS, MERS, common coronavirus, influenza, ADV, MPV, PIV, and as a function of DNA dots on microarrays found listed on the x-axis. It is a graph of the fluorescence score of IgG of RSV. [76] [78] Graphs of individual patient fluorescence scores for IgG and IgM detection in samples using microarrays and the corresponding Z-score statistics, the red line is an assessment of whether the measurement is positive or not. The blue line is the average of the negative results, the red corresponds to the mean serum reaction positive result further confirmed via PCR, and the blue line is confirmed via PCR. If the patient's IgG bar graph looks like a red line, the test is positive, if it looks like a blue line, the test is negative, and FIG. 18b is shown in FIG. 18d. Several viruses, namely SARS-CoV-2, SARS, MERS, common coronaviruses, influenza, ADV, MPV, PIV, and as a function of DNA dots on microarrays found listed on the x-axis. It is a graph of the fluorescence score of IgM of RSV. [76] [79] Graphs of individual patient fluorescence scores for IgG and IgM detection in samples using microarrays and the corresponding Z-score statistics, the red line is an assessment of whether the measurement is positive or not. The blue line is the average of the negative results, the red corresponds to the mean serum reaction positive result further confirmed via PCR, and the blue line is confirmed via PCR. If the patient's IgG bar graph looks like a red line, the test is positive, if it looks like a blue line, the test is negative, and Figure 18c is on the x-axis. IgG readings of several viruses, namely SARS-CoV-2, SARS, MERS, common coronavirus, influenza, ADV, MPV, PIV, and RSV, as a function of the DNA dots on the listed microarrays. It is a bar graph of Z score statistics. [76] [80] Graphs of individual patient fluorescence scores for IgG and IgM detection in samples using microarrays and the corresponding Z-score statistics, the red line is an assessment of whether the measurement is positive or not. The blue line is the average of the negative results, the red corresponds to the mean serum reaction positive result further confirmed via PCR, and the blue line is confirmed via PCR. If the patient's IgG bar graph looks like a red line, the test is positive, if it looks like a blue line, the test is negative, and Figure 18d is on the x-axis. IgM readings of several viruses, namely SARS-CoV-2, SARS, MERS, common coronavirus, influenza, ADV, MPV, PIV, and RSV, as a function of the DNA dots on the listed microarrays. It is a bar graph of Z score statistics. [81] Top transparent perspective of the Optician reader with the microfluidic bay exposed. [82] FIG. 6 is a side view of LED excitation for microarray assay and detection with CMOS cameras. [82] It is a transmission spectroscopic photograph of the excitation spectrum and the radiation spectrum of the sample in which the LED radiation filter and the camera notch filter spectrum are overlapped. [83] FIG. 3 is a block diagram of electronic components in Opticians used for microarray measurements. [84] It is a block diagram of an example of a cloud ecosystem that interfaces with a device, receives data, calculates results, and returns the results to the device. [85] FIG. 6 is a block diagram of a device having a SAW sensor as a detector. [86] FIG. 6 is a block diagram of a device having an optical sensor as a detector. [87] FIG. 6 is a diagram of disk CD-3 used for immuno-PCR showing a series of steps performed on an disk.

[88] The present disclosure and various embodiments thereof can be better understood by reference to the following detailed description of the preferred embodiments, the preferred embodiments being defined in the claims. It is presented as an example for the explanation of the embodiment. It is expressly understood that the embodiments defined by the claims may be broader than the embodiments for the purposes described below.

Detailed description of preferred embodiments
[89] Over the past few decades, an increasing number of emerging infectious diseases have caused serious social and economic consequences on a global scale. In particular, rural Third World communities are not only experiencing high exposure to infectious diseases, but also face many challenges in medical access. Nevertheless, pathogens do not identify borders, and outbreaks of new diseases affect people everywhere, wherever they are. Expert-selected knowledge, software, and services that support the interpretation of medical diagnostic test results from interconnected Point of Care (POC) networks around the world to track and prevent the rapid spread of infectious disease pandemics. , The only way humanity can be expected to maintain a vibrant economy and a highly fluid society.

[90] The disclosed approach of the illustrated embodiment first solves the problems associated with currently available COVID-19 diagnostic instruments by measuring both the pathogen directly and the pathogen antibody. SKC-Optilkus-2020 performs all three types of measurements listed above. Different types of tests require disposable cartridges of different types in the shape of compact discs or microfluidic discs (CDs), i.e. disc 29 CD-1 for ELISA, disc 31 CD-2 for immunofluorescence or SAW detection, And RT-qPCR require disc 26 CD-3. The protein array on Disc 29 CD-1 can be performed in less than 10 minutes. The time required for direct virus testing on Disc 31 CD-2 is less than 12 minutes. An antibody test on Disc 29 CD-1 is performed first, and if a pathogen antibody is found, a related pathogen test is performed (using either Disc 31 CD-2 or Disc 26 CD-3). ..

[91] Disc 29 The "multiplexed antibody array" on CD-1 provides an individual virus "exposure fingerprint", which is a "legacy antibody profile" that reflects past exposure and vaccination history. do. This array analysis technique is far more data-rich (eg, 67 antigens at 4 replications per array) and is more quantitative in current use for measuring antibodies against viruses. To understand this point, refer to FIG. 1 showing both positive and negative 2019 nCOV array sensitivity IgG results obtained from blood samples from the COVID-19 outbreak in Washington State, 2020.

[92] Second, the sample collection device 100 described in connection with FIGS. 13a-13f is directly coupled to the disposable compact disc. The sample preparation step is integrated on a fluid disk and cloud-based data processing is performed as described in connection with FIG.

[93] Includes bacteria, parasites, fungi, and viruses such as Wakusina, monkeypox, herpes 1 and 2, varicella-zoster, HPV, HIV, dengue, influenza, western Nile, chikungunya fever, adenovirus, and coronavirus. High-throughput cloning and construct microarrays 12 containing human and animal antibodies, along with antigens from more than 35 medically important pathogens, have been conventionally developed. A DNA microarray 12 (also commonly known as a DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. The DNA microarray 12 is used to simultaneously measure the expression levels of multiple genes or to genotype multiple regions of the genome. Each DNA spot contains a particular DNA sequence known as a probe (or reporter or oligo) of a few picomoles ( ^10-12 mol). These can be a short portion of a gene or cRNA, also called cDNA or antisense RNA, under high strict conditions, other DNA elements used for hybridization of samples called targets. Probe target hybridization is typically detected and quantified by detection of fluorophore, silver, or chemiluminescent labeled targets to determine the relative abundance of nucleic acid sequences in the target. The original nucleic acid array was a macroarray approximately 9 cm x 12 cm, and the first computerized image-based analysis was published in 1981. We probed over 25,000 samples from humans and animals infected with pathogens and identified over 1000 immunodominant and vaccine antigen candidates for these pathogens. We have individual proteins / antibodies printed on these arrays 12 capture antibodies and / or antigens present in serum from infected individuals and use fluorescent secondary antibodies. It was shown that the amount of antibody produced could be quantified.

[94] In this way, a comprehensive profile of antibodies that occur after infection or exposure can be identified that are specific to the type of infection and stage of the disease. Array 12 can be produced and probed in large numbers (> 500 serum or plasma per day), consuming less than 2 μl for each sample. This microarray approach allows the inspector to evaluate the antibody repertoire with a large collection of samples that is not possible with other techniques.

[95] A coronavirus antigen microarray 12 (COVAM) containing 67 antigens responsible for acute respiratory infections was constructed. The virus antigens printed on this array 12 are SARS-CoV-2, SARS-CoV, MERS-CoV, common cold coronaviruses (HKU1, OC43, NL63, 229E), and influenza, adenovirus, and metapneumovirus. , Parainfluenza, and / or from epidemic coronaviruses, including multiple subtypes of respiratory conjugation virus. The SARS-CoV-2 antigens in this array 12 are spike proteins (S), receptor binding (RBD), S1 and S2 domains, total protein (S1 + S2), and nucleocapsid proteins (S1 + S2), as shown in the graph of FIG. NP) is included. There is a similar set of antigens represented in the array from SARS-CoV, MERS-CoV, and four common cold coronaviruses.

[96] SARS-CoV-2 convalescent blood specimens from PCR-positive individuals (positive group) and collected prior to the COVID-19 pandemic from untested individuals to identify antibody profiles for SARS-CoV-2 infection. The degree of differential response to these antigens was evaluated for the serum (negative control group). As shown in the heatmaps of FIGS. 2a and 2b, the positive group is highly reactive to the SARS-CoV-2 antigen. This is more pronounced in the case of IgG than IgA. Negative controls do not respond to SARS-CoV-2, SARS-CoV, or MERS-CoV antigens, even though they are highly reactive to common cold coronavirus antigens. As shown in FIGS. 2a and 2b, the positive group showed high IgG reactivity to the SARS-CoV-2 NP, S2, and S1 + S2 antigens, and the degree of reactivity to SARS-CoV-2 S1. Lower. The positive group also showed high IgG cross-reactivity to SARS-CoV NP, MERS-CoV S2 and S1 + S2 antigens, while the negative group showed S1 + S2 and S2 antigens from SARS-CoV-2 and MERS-CoV. It shows low cross-reactivity with and does not show cross-reactivity with other SARS-CoV-2 antigens.

[97] Table 1 shows the Z-score statistics of the fluorescence intensity results with IgG shown in FIG. 18a, the fluorescence results in FIG. 18c, the fluorescence intensity results in IgM shown in FIG. 18b, and the fluorescence result Z in FIG. 18d. Includes score statistics. The Z-score indicates how many standard deviations the definite positive IgG or IgM sample has above or below the mean negative result. Statistically significant z-scores (5 and above) have shaded numbers.

[98] The antigen is then evaluated and a receiver operating characteristic (ROC) curve with measured area under the curve (AUC) is used to distinguish the positive group from the negative group over the entire assay cutoff value. High performance antigens that detect IgG are defined by ROC AUC> 0.85 as shown in Table 1. The four antigens are ranked as high performance antigens: SARS-CoV-2 NP, SARS-CoV NP, SARS-CoV-2 S1 + S2, and SARS-CoV-2_S2. Additional high performance antigens included SARS-CoV-2 S1 (with mouse Fc tag), RBD, and MERS-CoV S2. Optimal sensitivity and specificity were also estimated for the seven high performance antigens based on the Ioden index. The J-statistic of Yoden (also called the Yoden index) is one statistic that captures the performance of the dichotomous diagnostic test. Informedness is a generalization to multiclass cases and estimates the probability of informed decision. The lowest sensitivity is seen with SARS-CoV-2 S1 and correlates with the relatively low reactivity to this antigen in the positive group. The lowest specificity is found in SARS-CoV-2 S2 and correlates with the cross-reactivity of this antigen seen in a subset of the negative group. To estimate the performance gain from antigen binding, all possible combinations of up to 4 of the 7 high performance antigens were tested in silico for performance in distinguishing between positive and negative groups. ROC curves with AUC, sensitivity, and characteristics were calculated for each combination. There is a clear gain in performance by combining two or more antigens. For IgG, the best distinction was achieved using the two antigen combinations SARS-CoV-2 S2 and SARS-CoV NP, with similar performance when SARS-CoV-2 S1 was added with the mouse Fc tag. Was also achieved (AUC = 0.994, specificity = 1, sensitivity = 0.944). The addition of the fourth antigen reduced performance.

[99] Table 2 shows the performance data of the combination of high performance antigens. For each ranked individual antigen, the sensitivity and specificity based on the ROC, AUC value, and the Yoden index for the distinction between positive and negative sera are derived, and the high-performance antigen having ROC AUC> 0.86 is lined up. Shown above.

[100] FIGS. 18a-18d show an example of one confirmed positive patient result. FIG. 18a shows the normalized fluorescence intensities of various IgG antibodies in serum, and the two lines show the average results of confirmed positive (top) and confirmed negative (bottom). FIG. 18b shows the normalized fluorescence intensities of various IgM antibodies in serum, and the two lines show the average results of definite positive (top) and definite negative (bottom). FIG. 18c shows the plotted Z-scores of IgG antibodies between positive and negative results, with three dotted lines representing various low, medium, and high response Z-score thresholds. FIG. 18d shows the plotted Z-scores of IgM antibodies between positive and negative results, with three dotted lines representing various low, medium, and high response Z-score thresholds.

[101] By using the current COVID-19 pandemic as the most pressing target, it addresses some of the most pressing requirements to establish disease screening, interpretation, and prevention goals. Current COVID-19 detection platforms are divided into three categories: 1) enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, 2) real-time quantitative polymerase chain reaction (RT-qPCR), and 3) chest X. line. Point of care methods and devices are the focus of this disclosure and chest radiographs are not addressed herein.

[102]
[103] Optikus II
[104] We have used a compact disk (CD) microfluidic in a handheld instrument 10 to cartridge for a number of different assay targets as represented by the instrument 10 shown in the perspective view of FIG. Sample introduction (capillaries or cotton swabs), sample preparation (eg, weighing, diluting, blood plasma separation, and cell lysis), reagent storage, and SAW sensor 90 (for direct virus measurement) and fluorescent camera 89 (for protein assay measurement). ) Has been developed as a high-speed portable diagnostic screening device that automates quantitative measurements. FIG. 4 shows a microfluidic disk interface 11, a SAW RF shield 15, a multifunction button 17, an SD card 35, a USB port 19 including a Z-stage mobile platform 13 and a disk spindle motor 154, which are well described in the incorporated specification. , Speaker microphone 21, touch display 23, and status LED 33 are also shown.

[105] Optikus device 10 also performs RNA and DNA amplification directly on the microfluidic disk 26 (CD-3) using either thermal cycling or isothermal amplification, disk CD-1 shown in FIG. Also includes thermoelectric heating and cooling or temperature control mechanisms (not shown) in the Pelche element 142 and disk CD-1. The Optikus platform is unique in its ability to quickly adapt to new tests by modifying the microfluidic disk design and assay components. For example, Optikus II uses Disc 29 CD-1 (for ELISA detection using SAW) and Disc 31 CD-2 (for immunofluorescence detection) as described in the incorporated specification 2 Perform two COVIDEO-19 inspection sets. The development of a third cartridge (CD-3 for RT-qPCR) is also within the intended scope of the invention, as described herein. This specification primarily relates to the disclosure of a device 10 configured to perform immunofluorescence detection using a microarray 12 on disk 31 CD-2. Instrument 10 in FIG. 19 is the circuit and detector required to support or operate all three types of discs 26, 29, and 31 for ELISA detection, immunofluorescence detection, and RT-qPCR using SAW. Has.

[106] The Optikus II reader 10 includes various additional components for making measurements using different types of biosensors and microfluidic CDs. FIG. 19 shows an upper transparent perspective view of the Optikus II reader 10 with the microfluidic assembly bay 136 open, where the Optikus II reader 10 is a laser 138 that ablate various microvalves in the microfluidic CDCDs 26, 29, 31. Includes a SAW RF interposer 140 for RF measurements using the SAW detector 90, various Pelche elements 142 for adjusting the temperature on the disk, and a CMOS camera 89 for various fluorescence imaging measurements.

[107] FIG. 23 is a high level block diagram of equipment 10 in which the SAW reader 135 is coupled to the sensor interface 137 and provides an RF excitation signal to the SAW 90. Reader 135 includes a USB interface, power, serial data interface, display control and wireless communication. Control of the microfluidics on the disc CD-2 is controlled by the reader 135 through the microfluidic controller 133, which includes processor control of the laser and disc motor. The detailed operation of the device 10 in FIG. 23 is described in the referenced specification.

[108] FIG. 24 is a high level block diagram of the instrument 10 with the serological PCR reader 129 coupled to the camera 131. This high-level block diagram shows how a reader 129 with a camera 131 works as both a camera for a serological solution and a reader for a chemical luminescent PCR solution. Reader 129 can perform both PCR and serology. The reader 129 in the illustrated embodiment is based on a Rasberry PI single board computer and includes a USB interface, power, serial data interface, display control / wireless communication. The control of the microfluidics in the disk 26 CD-3 is controlled by the reader 129 through the serological microfluidic controller 127, which is the thermoelectric (TEC) heating of the laser, disk motor, and Pelche element 142. And processor control of cooling control. The detailed operation of the device 10 in FIG. 23 is described in the referenced specification.

[109] FIG. 25 is a diagram of the immuno-PCR steps performed automatically on Disc 26 CD-3 and Disc 26 CD-3. Similar steps are detailed in the incorporated specification. The blood sample collected by pointing a finger in step 65 is placed in the sample chamber 67. In blood serum separation step 61, blood and serum are automatically separated in the blood-serum separation chamber 63. In step 57, the serum is transferred to the mixing chamber 59, mixed and conjugated with the antibody DNA complex that is the target 53. The conjugated target 53 is transferred to a mixing / replication chamber 55, where it is mixed and conjugated with an antibody-magnetic nanoparticles (MNP) complex with a DNA tag, as fully described in the incorporated specification. In step 47, plasma is removed by transfer to plasma disposal chamber 49. In step 46, the buffer sent from the wash reservoir 45 through chamber 59 to chamber 55 removes unconjugated DNA. In step 77, the conjugated antibody target complex is resuspended in the amplification buffer supplied from the amplification buffer chamber 79 to the chamber 55. Next, in step 81, isothermal amplification is performed in chamber 55 during replication or PCR. Then, in step 103, the replicated DNA tag is transferred to the imaging chamber 87 and bound. If necessary, optional washing and spin drying steps 105 may be performed to remove the unbound DNA tags into the waste chamber 107 to improve imaging results. Additional backup details for the method shown in FIG. 25 are provided in the incorporated specification.

[110] FIG. 21 is a block diagram of a circuit used in the microarray reader 134 of the Optician device 10 to read disk 29 CD-1. A similar block diagram of the circuit used in the reader utilizing the SAW90 as a detector is detailed in the referenced specification. The disc 29 is shown graphically as including a loading chamber 70 that transfers the sample to the blood-plasma separation chamber 72. Magnetically tagged elements are detected by light and magnetic fields acted upon by magnets / cavities 153 and controlled through a magnetic / optical index drive 157 coupled to microprocessor 148. The magnet 153 acts as an indexer so that the position of the disk 29 can be checked by the reader 134 each time the magnet 153 on the disk 29 passes through a magnetic sensor (not shown). The disk position is then compared to the index on the motor 154 and modifications are applied if necessary. Therefore, an off-motor indexer can be realized by using an optical sensor and a reflection sticker or a magnet and a magnetic field sensor on the disk 29.

[111] When the separated plasma is transferred from the chamber 72, the plasma is measured by the light detection circuit 71 coupled to the LED drive 73, and the returned signal is amplified by the op amp 75, the circuits 73 and 75. Are both coupled to microprocessor 148 and controlled by microprocessor 148. The laser 138 used to selectively open the valves on the disk 29 is controlled through the laser drive 139 and the op amp 141, both controlled by the microprocessor 148. The treated sample is transferred to the reaction detection chamber 76 on the disk 29, where it reacts with the DNA dots on the microarray 12. The camera 164 takes a color picture of the microarray 12 and the image is communicated to the microprocessor 146 via the image signal processor 166. Under program control stored in RAM memory 147 and flash memory 149, the clocked processor 146 formats the microarray data and communicates to the cloud service 106 through the wireless module 168, as further described in connection with FIG. do.

[112] The reader 134 is controlled by two microprocessors 146 and 148, one processor 146 arranged on an off-the-shelf (OTS) mainboard 150 and one processor 148 arranged on a microfluidic board 152. The microprocessor 148 on the microfluidic substrate 152 controls the spindle motor 154 using an encoder through the motor drive 155, controls the geared motor using the encoder driven by the motor drive 159, and couples through the low pass filter 161. Controlled by the limit switch 158, the motor 156 rotates the disk 29 so that it can be placed in a selected or controlled angular position for measurement. The microfluidic substrate 152 controls the IR LED 160 through the LED drive 162 to excite the fluorophore. The CMOS camera 89 is controlled by the main board OTS150 through the image signal processor 166. The main board OTS150 controls a Wi-Fi module 168, a Bluetooth module (not shown), a digital display 170, and a power interface 172. The microprocessor 148, which operates under program control stored in the memory 151, uses the temperature / humidity sensor 145 to adjust the environment-dependent operation in the disk 29.

[113] Similar schematics for use with Disc 26 CD-3 and Disc 31 CD-2 are included in the Appendix and are not discussed further here.

[114]
[115] Assay-Protein Assay
[116] The protein microarray 12 utilizes the probe of Optician II equipment 10 to analyze the target and quantify the antigen-specific antibody reaction induced after infection from any microorganism. In array 12, approximately 70,000 proteins from 35 infectious agents were probed and analyzed using DNA spots printed on protein microarray 12. Array 12 was probed with thousands of serum specimens from infectious disease cases and controls to identify specific antigens that induce antibodies after infection. This array 12 in particular understands who was exposed to the virus in the environment, predicts who is suspected of becoming severe, who is mildly infected, who is asymptomatic, and who is exposed and protective. The urgent need to identify who is unknowingly spreading the infection to close contacts is directly linked to today's problems associated with coronavirus outbreaks. This type of serum survey data can find "hot spots" where infectious agents are present in the local population and public health mitigation and containment measures should be collected.

[117] A currently open benchtop workflow with four steps is shown in FIG. 5: 1) microarray print step 14, 2) probing step 16, 3) imaging step 18, and 4) analysis step 20. Microarray 12 is used to quantify and identify post-infection-induced Ab reactions. Currently, the capacity of each step identified in FIG. 5 is as follows. The print step 14 uses a conventional protein microarray printing facility capable of printing 1.4 million protein antigen spots 22 per day on 4,800 microarrays. Probing step 16 is a benchtop probing method capable of probing 50,000 serum specimens per day on a microarray 12 by conjugating specific fluorescent tags 24, 27 to the corresponding antibodies 23, 25. The quantification or imaging step 18 uses a robotic laser scanner to quantify the level of Ab bound to the antigen printed on the array 12. Analysis step 20 uses software to help organize and interpret the results 28 of the inspection of array 12 (see examples in FIGS. 1a and 1b).

[118] Assay modification steps for use on CD
[119] A small antigen-antibody array containing antigens or antibodies from or against the following viruses to adapt the microarray assay described in connection with FIG. 5 to the CD-based fluid engineering platform of Instrument 10. Twelve are prepared: SARS-CoV-2, SARS-CoV-1, MERS, influenza, adenovirus, parainfluenza, metapneumovirus, and respiratory conjugate virus. The entire probing process takes about 15 minutes. Briefly, 10 μl of plasma for detecting the antibody on the antigen array 12 or a swab sample for the nose for detecting the viral antigen on the antibody array 12 was dried in step 32 of FIG. preblocked array) 12 is placed directly. After culturing for 5 minutes, in step 34, 10 μl of the fluorescently labeled secondary Ab is added while extruding the sample from the array chamber. In step 36, the array 12 is washed, centrifuged and dried in step 38, and then in step 40, the plasma sample is ready to capture images of up to three colors using a fluorescent camera for different Ig isotypes. .. The image is then sent to the cloud for analysis.

[120] FIG. 20a is a diagram showing fluorescence measurement of the microarray 12 performed on the disk 29 using the CMOS fluorescence camera 89. The LED 91 emits fluorescence-excited photons, which pass through a preliminary synchrotron radiation filter 84 with a bandpass peak of 750 nm. The filtered excitation photons interact with the microarray 12 mounted on the nitrocellulose membrane 85 carrying the fluorofore microarray 12. The sample interacts with the microarray 12 in the reaction chamber 56. The excited or induced emitted fluorescent photons then pass through a camera notch filter 83 centered around 790 nm and then collected by a CMOS color camera 89 for processing and communication to the cloud service 106 on a microarray reader. Sent to 134.

[121] FIG. 20b shows the absorption of the fluorophore or excitation spectrum 41 and the spectrum graph of the emission or emission spectrum 43 of the fluorophore. The bandpass of the LED radiation filter 84 and the notch filter 83 is superimposed on the excitation spectrum 41 and the radiation spectrum 43. The filter 84 confine the excitation light from the LED 91 to a wavelength range that overlaps the lower half of the fluorofore excitation spectrum of the DNA spot 22 on the microarray 12. The notch filter 83 limits the wavelength received by the camera 89 to the larger domain of the emission spectrum 43 without completely overlapping the bandpass of the filter 84. Therefore, the color image generated by the camera 89 is only that of the excited fluorophore in the DNA spot 22 of the microarray 12, and is not an image of any of the excited light from the laser 91.

[122] Further, the figure shows the existence of two cutoff filters that demarcate the boundaries between LED excitation and CMOS camera detection.

[123] Alternatively, the array 12 can also be adapted to perform the neutralizing antibody assay shown graphically in FIG. 7. The array 12 spotted with the receptor binding domain (RBD) of the peplomer is probed with a plasma sample from the patient and a fluorofore-labeled ACE2 (eg, Alexa Fluor 488) in step 42 and the sample in the array chamber in step 44. While extruding from, another fluorophore (eg, Alexa Fluor 647) -labeled secondary antibody against human IgG (to detect RBD antibody in the sample) is added. The array 12 is then washed in step 6 and centrifuged and dried in step 48, ready for image capture of each of the two colors of the RBD antibody and ACE2 present in the patient sample in step 50. Samples without neutralizing antibody or RBD antibody are detected using ACE2 fluorescence, while samples with a large amount of neutralizing antibody that have RBD antibody or interfere with ACE2-RBD binding have a reduced amount of ACE2 fluorescence. Is detected using. In the absence of RBD antibody from the plasma sample, the amount of ACE2 fluorescence can be quantified for relative neutralizing activity. Plasma samples can be applied directly to the CD platform and processed for image acquisition. This simplifies the design of fluid engineering systems.

[124]
[125] Adaptation of protein array assays to deployment on CD-1
[126] While the techniques shown in FIGS. 5-7 work well in laboratory settings, there are some limitations that impede deployment in a wider field. For example, the use of open wells is clearly not possible in POC settings, and the cost of laser scanners is also exorbitant in such settings. Instead, we have developed a 10-minute automatic POC coronavirus antigen microarray containing one array per disposable fluid engineering disc CD-1 cartridge. A digital fluorescence microscope is used for quantification. To implement this scenario, the protein array print remains the same as above, but here the methodology must be implemented in large-scale commercial expansion production.

[127]
[128] Array disconnection
[129] Multiple arrays 12 are typically manufactured on a substrate in a batch process, but one array 12 is used for each cartridge (disk CD-1) mounted on the corresponding nitrocellulose thin film slide. Array 12 integrated into disk CD-1 simultaneously probe 60 antigens from 12 known coronaviruses along with several types of adenovirus, RSV, metapneumovirus, parainfluenza, and influenza virus. This test can reveal IgG, IgA, and IgM seroreactivity to different viruses and is useful in identifying seroprevalence of SARS-CoV-2.

[130]
[131] CD Fluid Engineering
[132] FIGS. 8a and 8b show a CD system for low cost, high throughput automated immunoassay processing. The disposable immunoassay disc design is such that the centrifugal acceleration acting on the liquid element first produces and stores air energy, which is then released by the decrease in centrifugal acceleration, resulting in the opposite direction of fluid flow. Includes fluid engineering structures that allow highly efficient micromixing based on reciprocating mechanisms. Through an alternating sequence of high and low centrifugation acceleration during the culture / hybridization phase of the assay, the system reciprocates a flow of liquid within the disc to culture / hybridize efficiency between the antibody and antigen micromolecules. To maximize. A schematic diagram of the fluid engineering system 52 and the antibody capture 54 in each array element is shown in FIG. 8a. The reaction chamber 56 in FIG. 8a holds the spotted protein array 12. The sample is loaded into the loading chamber 58 of the CD and transferred to the upper chamber 60 through centrifugal force. The sample is also transferred through the duct 66 to the reaction chamber 56, where it is probed by the array 12. The surplus sample is transferred to the siphon 62 and discarded in the waste chamber 64.

[133] For proof of concept, an immunoscreening experiment was prepared by creating an array of Burkholderia antigens and probing with infected and uninfected sera. The characteristic enzyme of the sample, which is colorless in solution, is conjugated to the antigen captured in array 12 via the secondary Ab and the primary Ab. The product fluoresces as a dark blue precipitate. Burkholderia is a bacterial pathogen that attacks the human respiratory system and causes melioidosis. Symptoms can include chest, bone, or joint pain, cough, skin infections, and lung nodules.

[134] The main concerns driving the miniaturization and automation of immunoassays involve high cost reagents such as antibodies and antigens, high cost labor force, and long assay time. Based on the results of the proof-of-concept, we used the described reciprocating fluid engineering system to reduce reagent consumption by about 75% and assay time by about 85% for immunoassays. Demonstrated that it was possible to do. See FIG.

[135] The system implemented on Disc CD-1 can use (1) blood samples, (2) fluorescence instead of absorption measurements, thereby eliminating the need for timing and (3). It is simplified because the quantification is performed using an inexpensive digital fluorescence microscope.

[136] FIGS. 10a to 10c are graphs showing simulation results. FIG. 10a compares three culture modes: single flow through, reciprocating flow, and passive diffusion between human IgG antigen and goat anti-human IgG antibody. FIG. 10b shows the effect of antibody enrichment on the rate of complex formation. As the initial enrichment of the sample decreases, the antigen-antibody complex formation decreases over time and reaches an equilibrium state. FIG. 10c shows the effect of the Flow Reynolds number on the complex formation rate. As the flow rate increases, antigen-antibody complex formation increases and reaches equilibrium. This difference is more pronounced for antibodies with lower initial enrichment.

[137] In addition to reciprocating motion, other fluid engineering functions performed by us on Disc CD-1 are 1) sample weighing, 2) blood plasma separation, and 3) fluorescence detection. In FIG. 11a, these functions are shown with the protein array 12 as well as a CMOS chip containing a laser diode and a CCD detector under the CD for signal readout. The sample is loaded into the sample loading chamber 70 of the CD and transferred to the blood plasma separation chamber 72 by centrifugal force. Target-carrying plasma is transferred to reaction detection chamber 76 via siphon 74, which can optionally communicate with valved wash buffer from chamber 78, increasing mass from chamber 80. Therefore, the air pocket of the chamber 82 can be used for reciprocating motion to optionally couple with gold nanoparticles. Detection is achieved by the use of a 650 nm diode laser directed at the array 12 in chamber 76, read by a Si-CCD array positioned under the CD.

[138] FIG. 12 shows how a blood sample is obtained from pointing a finger and collected in a 300 μL Microbette 88 (CB300). The blood droplet 39 is pulled out by a finger puncture by a nested microcapillar 37 and enters the cavity 71. The microcapillaries 37 are nested in a microcubette 88 sealed with a cavity 71 and a cap 68. When the sample should be sent to the disc 26, 29, or 31, the microcapillaries 37 are nested and the blood from the cavity 71 is transferred to the sample loading chamber 70 of the CD rotor 86 via the microcapillaries 37 by capillary action. So the first step, as mentioned above, includes sample volume weighing and blood plasma separation.

[139]
[140] Direct COVID-19 assay adaptation to deployment on CD-2
[141] Disc 29 If the results on CD-1 are positive for the presence of viral antibodies, then CD-2 disc 31 is fast (less than 12 minutes) for detecting COVID-19 directly from the nasal swab sample. Used for point of care diagnostic tests. CD-2 uses the surface acoustic wave biosensor 90 (SAW) directly for COVID-19 detection. Traditionally, the SKC SAW sensor 90 has succeeded in detecting multiple high profile bacteria and viruses, including Ebola and B. anthracis. Over the last two years, we have significantly improved the sensitivity and detection capabilities of the SAW biosensor 90. The sample introduction from the cotton swab tip 95 to the CD31 is shown in FIGS. 13a to 13f, and the SAW sensor 90 mounted on the SKC disk 31 is shown in FIG. In the embodiment of FIG. 14, the disk 31 includes two SAW sensors 90 along with a fuzz button connector 101.

[142] FIGS. 13a and 13b are cross-sectional views of the conical device 100 inserted into the disc 31 before the disc 31 is inserted into the device 10. The conical device receives the nasal swab 94, cuts it, resuspends it in buffer / lysing beads 98, releases reagent 93, performs cytolysis, and retracts the sample to disk 31. The reagent pouch 92 is a chamber containing the reagents selected for handling the sample from the swab 94. The swab tip 95 carries the target sample, is fully inserted into the swab chamber 102 as shown in FIG. 13a, and slides past the downward tilt blade 96 without being cut. When the swab shaft 94 is pulled upward, the swab shaft 94 is forcibly pressed against the downward tilting blade 96 to be cut, and when the swab shaft 94 is removed from the device 100, the swab shaft 94 is as shown in FIG. 13b. Allow the tip to stay at the bottom of the device 100. As shown in FIG. 13c, the sealing cap 104 is pushed down to rupture and open the buffer pouch 92, allowing the reagent or buffer 93 to flow into the swab chamber 102 through the duct 97 and into the cut swab tip 95. Make contact. As shown in FIG. 13d, the sealing cap 104 is fully depressed to seal the inlet to the device 102. As shown in FIG. 13e, the device 100 is manually angularly vibrated to dissolve the sample target. Free metal beads 98 are present in the swab chamber 102 to assist in dissolution during agitation. The device 100 is then manually rotated to retract from the swab chamber 102 through the peripheral opening as indicated by arrow 99 and transfer the dissolved target sample as shown in FIG. 13f.

[143]
[144] SKC Optikus Cloud Platform
[145] As an Internet of Things (IoT) device, the Optician II device 10 provides critical data for the diagnosis of illness and disease. Optikus II measurements are transmitted to the cloud service 106 over an encrypted secure HTTPS link using the device API 108, which is the local Optikus cloud, as shown in FIG. Cloud Service 106 is a microservices API platform that currently uses best practices for security, resiliency, and reliability. This allows authorized access to test results from hospital 112, doctor 114, and patient 110 itself. The test results are stored in the Oracle database 116 for deep learning analysis and in the distributed ledger technology 118 (DLT) or blockchain database to ensure data transparency, high availability, and immutability.

[146] FIG. 16 shows a step of processing image data output from a digital fluorescence imaging capable Optician II device 10 used in combination with cloud-based data processing. In step 120, as described above, the sample is taken from the patient in the field and probed in device 10. The image of the microarray 12 is acquired by the device 10 in step 122 and uploaded to the cloud server 106 in the cloud in step 124. In step 126, the detected antigen or antibody is quantified from the uploaded data and in step 128 undergoes further data processing. The result data is then provided for individual patient and public health data analysis in step 130. The data and its analysis are then provided for clinical trials in step 132, i.e. integrated with Oracle Cloud Network 106 to compile all clinical trial data, whereby the analyzed data is the point of care site. Will be provided immediately.

[147] FIG. 22 is a more detailed view of the cloud ecosystem 176, 178, 180, in which the device 10 interfaces with the user mobile phone 174, receives microarray data, calculates diagnostic results, and returns diagnostic analysis to mobile phone 174. It is a figure. In the illustrated embodiment, a user or patient seeking a COVID-19 test using telephone 174 registers online with an Oracle data management system (DMS) 176 through registration module 182. The user identifies, module 184 schedules one or more specified inspections at a specified date and time and at a location near the user, and all relevant data for the user is stored in database 186. A unique Quick Response (QR) code® is assigned to the patient examination event and transmitted via module 188 to the user's telephone 174 as a short message service (SMS).

[148] The user visits the inspection site 180 at the scheduled appointment, identifies himself using the assigned QR code®, the check-in event is processed by module 190, and the user in database 186. Accumulated in the data record. Data associated with a QR code® that identifies the patient and one or more tests to be performed is read or scanned by the device 10 at the test site 180. In step 194, the inspection is performed using the device 10 as described above. After sampling, all steps in the ecosystem 176, 178, 180 are automatic and are performed sequentially under software control without the need for further human intervention. The assay is performed, assay data is uploaded, analyzed, stored, diagnosed and the results are reported to the patient within an hour, usually within tens of minutes. In step 196, a color photographic microassay record is created as a tagged image file format (TIFF) or other graphical format file. In step 198, the color photographic microassay record is packaged with the QR scan code by the instrument 10 and transmitted to the inspection service cloud site 178, and in step 200, it is received wirelessly at the inspection service cloud site 178 and the inspection service cloud site 178. It is uploaded to the database 202 of. The assay results are processed, diagnosed and converted into JavaScript object notation (JSON) based on the test generated in step 204. JSON is a data exchange format that stores and transmits data objects of attribute-value pairs and array data types (or any other serializable value) using an open standard file format and human readable text. This is a very common data format and has a wide variety of uses such as acting as an alternative to XML in AJAX systems. The resulting JSON and TIFF files are communicated over the Internet in step 206 and stored in object storage 208 in Oracle DMS176, from which they are stored in database 186 and inserted into the patient's record. Next, the inertial result is communicated from the result portal 210 to the user's telephone 174. The entire process runs automatically in 30-60 minutes or less. Benchmark events are automatically shared between the user's phone 174 and Oracle DMS176 along with a medical partner (HCP) phone 212 that may perform or initiate medical intervention as needed. If further diagnostic steps are desired or public health reports and responses are required, further processing is performed in connection with the inspection cloud service 178 through module 214 and independently by Oracle cloud DMS176 through module 216. Ru.

[149] Many modifications and changes may be made by one of ordinary skill in the art without departing from the spirit and scope of the embodiment. Therefore, it must be understood that the illustrated embodiments are described for purposes of illustration only and should not be construed as a limitation of the embodiments defined by the following embodiments and various embodiments.

[150] It should therefore be understood that the illustrated embodiments are described for illustrative purposes only and should not be construed as a limitation of the embodiments defined by the claims below. For example, even though the elements of the claim are described below in a particular combination, the embodiment claims a smaller number, more, or other combination of different elements than described above, first in such a combination. It must be explicitly understood to include, even if it is not listed in. The teaching that two elements are combined in the combination described in the claim may be used in the combination described in the claim, where the two elements are not combined with each other and may be used alone or in combination of other combinations. Please be further understood as possible. The deletion of any disclosed element of the embodiment is expressly intended to be within the scope of the embodiment.

[151] The terms used herein to describe various embodiments cover not only the commonly defined meanings, but also the scope of the commonly defined meanings by the special definitions herein. It should be understood to include structures, materials, or behaviors that go beyond. Therefore, where an element can be understood in the context of this specification as having more than one meaning, its use in a claim is all possible meanings supported by this specification and the term itself. Must be understood as a generic term for.

[152] Therefore, the following definitions of terms or elements in the scope of claims perform not only literally the combinations of elements described herein, but also substantially the same function in substantially the same manner. Is defined as including all uniform structures, materials, or operations that yield substantially the same result. Thus, in this sense, even replacement of two or more elements may also be made with any one of the elements in the claims below, or one element replaces the two or more elements in the claim. Is intended to be. The elements may be described above as operating in a particular combination, and may even be first described as such, but in some cases may include one or more elements from the combination described in the claim. It should be expressly understood that a combination can be removed from a combination and the combination described in the claim can be directed to a sub-combination or a variant of the sub-combination.

[153] Subtle changes from the intent described in the claims seen by one of ordinary skill in the art, which are currently known or later devised, are expressly intended to be equally within the claims. To. Therefore, obvious substitutions now or later known to those of skill in the art are defined as being within the defined elements.

[154] Therefore, the scope of claims includes, in particular, those shown and described above, those that are conceptually equivalent, those that are clearly substitutable, and those that fundamentally incorporate the basic concepts of embodiments. Please understand.

10 Handheld equipment 11 Microfluid disk interface 12 Microarray 13 Z stage moving platform 14 Print step 15 SAW RF shield 16 Probing step 17 Multi-function button 18 Imaging step 19 USB port 20 Analysis step 21 Speaker microphone 22 Protein antigen spot 23 Touch display 24 , 27 Fluorescent Tag 25 Antibody 26, 29, 31 Disc 28 Result 33 Status LED
35 SD card 37 Nested microcapsule 41 Excitation spectrum 43 Radiation spectrum 45 Wash reservoir 49 Plasma disposal chamber 52 Fluid engineering system 53 Target 54 Antibody capture 55 Mixing / replication chamber 56 Reaction chamber 59 Mixing chamber 60 Upper chamber 62, 74 Siphon 63 Blood Serum Separation Chamber 64 Waste Chamber 66 Duct 67 Sample Chamber 68 Cap 70 Loading Chamber 71 Cavity 72 Blood Plasma Separation Chamber 73 LED Drive 75, 141 Optoelectronic 76 Reaction Detection Chamber 78, 80, 82 Chamber 79 Amplification Buffer Chamber 83 Camera Notch Filter 84 Preliminary radiation filter 85 Nitrocellulose membrane 87 Imaging chamber 88 Microbette 89 Fluorescent camera 90 SAW sensor 91 LED
92 Reagent Pouch 93 Reagent 94 Nasal Swab 95 Swab Tip 96 Downward Tilt Blade 98 Buffer / Dissolving Bead 99 Arrow 100 Conical Device 101 Fuzz Button Connector 102 Swab Chamber 104 Sealing Cap 106 Cloud Server 107 Waste Chamber 108 Device API
110 Patient 112 Hospital 114 Doctor 116 Oracle Database 118 Distributed Ledger Technology 129 Serological PCR Reader 131, 164 Camera 133 Microfluidic Controller 134 Microarray Reader 135 SAW Reader 136 Microfluidic Assembly Bay 137 Sensor Interface 138 Laser 139 Laser Driver 140 SAW RF Interposer 142 Perche Element 145 Temperature / Humidity Sensor 146 148 Microprocessor 147 RAM Memory 149 Flash Memory 150 Ready-made Main Board 151 Memory 152 Micro Fluid Board 153 Magnet / Cavity 154 Disk Spindle Motor 155 159 Motor Drive 156 Motor 157 Magnetic Optical index drive 158 limit switch 160 IR LED
161 Low Pass Filter 162 LED Drive 164 Camera 166 Image Signal Processor 168 Wireless Module 170 Digital Display 172 Power Interface 174 Mobile Phone 176 Cloud Ecosystem 178 Inspection Service Cloud Site 180 Inspection Site 182 Registration Module 184, 188, 190 Module 186, 202 Database 208 Storage 210 Results Portal 212 Medical Partner Phone

[59] A graph showing IgG serum reactivity as measured by the fluorescence intensity of serum specimens on a coronavirus antigen microarray relative to frequency, the array being performed on disk CD-1 on the Optikus platform, FIG. 1a. Fluorescence spectrophotographic segments corresponding to multiple viruses shown side by side. [59] A graph showing IgG serum reactivity as measured by the fluorescence intensity of serum specimens on a coronavirus antigen microarray relative to frequency, the array being performed on disk CD-1 on the Optikus platform, FIG. 1b. Expansion of the spectrum of COVID-19 protein, SARS-CoV protein, and MERS-COVID protein. [60] A heatmap of coronavirus antigen microarrays, which are organized into columns classified as positive or red (recovery from PCR-positive individuals) or negative or blue (from uninfected individuals before pandemics). The serum showed IgG reactivity in FIG. 2a and IgA reactivity in FIG. 2b measured as average fluorescence intensity over four replications matched against each DNA dot in a microarray organized in virus-colored rows, showing reactivity. Is presented in color below the heatmap (white = low, black = medium, red = high). [61] Normalized IgG reactivity of positive and negative sera on coronavirus antigen microarrays, plots from convalescent sera (positive, red) from PCR-positive individuals and uninfected individuals before pandemics. The heat map below the plot shows the IgG reactivity for each antigen measured as mean fluorescence intensity (MFI) with full range (bar) and quadrant (box) in serum (negative, blue). The average reactivity of each group is shown (white = low, black = medium, red = high), and the antigen labeling is colored in each virus group. [62] A perspective partial transparent view of Optikus II that processes viral samples to quantify the antigen-specific antibody response induced after infection, and the sample preparation step-depending on the assay-is sample introduction, blood plasma. Includes isolation, plasma isolation, amplification or culture, washing, and measurement (SAW detection for viruses and fluorescence intensity detection for antibodies). [63] FIG. 6 illustrates an assay procedure on a microarray performed on a laboratory benchtop. [64] It is a figure which shows the method of the antiviral Ab detection in a blood using an antigen array, or the virus antigen detection in a nasal discharge by an antibody array. [65] FIG. 6 is a diagram of a method of using an antibody neutralizing assay using an array of RBD antigens. [66] It is a schematic diagram of the fluid engineering system in the disk CD-1. [66] Schematic representation of antibody capture at each array element, the reaction chamber holds a spotted protein array. [67] In a graph of experimental results comparing the strength of an IgG microarray made using a reciprocating flow system with different culture times with the strength of an IgG microarray made using a manual method with a culture time of 1 hour. be. [68] Compare the characteristics of reciprocating flow in the illustrated embodiments of culture compared to single flow and passive diffusion. [68] Compare the characteristics of reciprocating flow in the illustrated embodiments of culture compared to single flow and passive diffusion. [68] Compare the characteristics of reciprocating flow in the illustrated embodiments of culture compared to single flow and passive diffusion. [69] FIG. 6 is a diagram of a fluorescent biosensor of an illustrated embodiment of a protein array having a CMOS laser / diode chip underneath for signal readout on disk CD-1. [70] FIG. 6 shows a microlancet and a 300 μL microbette into which a blood sample has been drawn. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [71] In a cross-sectional view of a cone that can accept a nasal swab, cut it, resuspend it in buffer / lysed beads, release reagents, perform cytolysis, and retract the sample to disk. be. [72] FIG. 6 is a diagram of a CD rotor containing two SAW detectors corresponding to the fluid engineering circuit and RF interposer on disc CD-2. [73] Optician II cloud infrastructure diagram. [74] It is a figure which shows the usage of Optician II using a cloud infrastructure. [75] It is a diagram of the structure of a novel coronavirus. [76] [77] Graphs of individual patient fluorescence scores for IgG and IgM detection in samples using microarrays and the corresponding Z-score statistics, the red line is an assessment of whether the measurement is positive or not. The blue line is the average of the negative results, the red corresponds to the average serologic positive result further confirmed via PCR, and the blue line is confirmed via PCR. If the patient's IgG bar graph looks like a red line, the test is positive, if it looks like a blue line, the test is negative, and FIG. 18a is shown in FIG. 18c. Several viruses, namely SARS-CoV-2, SARS, MERS, common coronavirus, influenza, ADV, MPV, PIV, and as a function of DNA dots on microarrays found listed on the x-axis. It is a graph of the fluorescence score of IgG of RSV. [76] [78] Graphs of individual patient fluorescence scores for IgG and IgM detection in samples using microarrays and the corresponding Z-score statistics, the red line is an assessment of whether the measurement is positive or not. The blue line is the average of the negative results, the red corresponds to the mean serum reaction positive result further confirmed via PCR, and the blue line is confirmed via PCR. If the patient's IgG bar graph looks like a red line, the test is positive, if it looks like a blue line, the test is negative, and FIG. 18b is shown in FIG. 18d. Several viruses, namely SARS-CoV-2, SARS, MERS, common coronaviruses, influenza, ADV, MPV, PIV, and as a function of DNA dots on microarrays found listed on the x-axis. It is a graph of the fluorescence score of IgM of RSV. [76] [79] Graphs of individual patient fluorescence scores for IgG and IgM detection in samples using microarrays and the corresponding Z-score statistics, the red line is an assessment of whether the measurement is positive or not. The blue line is the average of the negative results, the red corresponds to the mean serum reaction positive result further confirmed via PCR, and the blue line is confirmed via PCR. If the patient's IgG bar graph looks like a red line, the test is positive, if it looks like a blue line, the test is negative, and Figure 18c is on the x-axis. IgG readings of several viruses, namely SARS-CoV-2, SARS, MERS, common coronavirus, influenza, ADV, MPV, PIV, and RSV, as a function of the DNA dots on the listed microarrays. It is a bar graph of Z score statistics. [76] [80] Graphs of individual patient fluorescence scores for IgG and IgM detection in samples using microarrays and the corresponding Z-score statistics, the red line is an assessment of whether the measurement is positive or not. The blue line is the average of the negative results, the red corresponds to the mean serum reaction positive result further confirmed via PCR, and the blue line is confirmed via PCR. If the patient's IgG bar graph looks like a red line, the test is positive, if it looks like a blue line, the test is negative, and Figure 18d is on the x-axis. IgM readings of several viruses, namely SARS-CoV-2, SARS, MERS, common coronavirus, influenza, ADV, MPV, PIV, and RSV, as a function of the DNA dots on the listed microarrays. It is a bar graph of Z score statistics. [81] Top transparent perspective of the Optician reader with the microfluidic bay exposed. [82] FIG. 6 is a side view of LED excitation for microarray assay and detection with CMOS cameras. [82] It is a transmission spectrophotograph of the excitation spectrum and the radiation spectrum of the sample in which the LED radiation filter and the camera notch filter spectrum are overlapped. [83] FIG. 3 is a block diagram of electronic components in Opticians used for microarray measurements. [83] FIG. 3 is a block diagram of electronic components in Opticians used for microarray measurements. [83] FIG. 3 is a block diagram of electronic components in Opticians used for microarray measurements. [84] It is a block diagram of an example of a cloud ecosystem that interfaces with a device, receives data, calculates results, and returns the results to the device. [85] FIG. 6 is a block diagram of a device having a SAW sensor as a detector. [86] FIG. 6 is a block diagram of a device having an optical sensor as a detector. [87] FIG. 6 is a diagram of disk CD-3 used for immuno-PCR showing a series of steps performed on an disk.

[110] FIGS. 21a- 21c are block diagrams of the circuit used in the microarray reader 134 of the Optician device 10 to read the disc 29 CD-1. A similar block diagram of the circuit used in the reader utilizing the SAW90 as a detector is detailed in the referenced specification. The disc 29 is shown graphically as including a loading chamber 70 that transfers the sample to the blood-plasma separation chamber 72. Magnetically tagged elements are detected by light and magnetic fields acted upon by magnets / cavities 153 and controlled through a magnetic / optical index drive 157 coupled to microprocessor 148. The magnet 153 acts as an indexer so that the position of the disk 29 can be checked by the reader 134 each time the magnet 153 on the disk 29 passes through a magnetic sensor (not shown). The disk position is then compared to the index on the motor 154 and modifications are applied if necessary. Therefore, an off-motor indexer can be realized by using an optical sensor and a reflection sticker or a magnet and a magnetic field sensor on the disk 29.

Claims (21)

An automated method of a cloud-based ecosystem that determines the presence of viral antigens and / or antibodies by field diagnostic testing of samples taken from subjects in portable handheld devices.
Placing the sample in the receiving chamber of the rotatable disk of the device and
The sample on the rotatable disk using the device according to the nature of the sample under automatic control and using the corresponding detection means in the portable handheld device for the viral antigen and / or antibody to be diagnostically tested. To handle and
To detect the quantitative measurement of the viral antigen and / or antibody in the sample using the corresponding detection means in the portable handheld device under automatic control.
To generate a data output of the detected quantitative measurement of the viral antigen and / or antibody in the sample corresponding to the subject under automatic control.
Communicating the data output corresponding to the subject to the cloud-based database under automatic control.
In a cloud-based ecosystem under automated control, the subject has the highest or previous probability of having multiple different types of viral antigens and / or antibodies and the communicated data output corresponding to the subject. If you have a virus infection that you are most likely to have, diagnose the type of virus infection and
Communicating the results of the comparative analysis from the cloud-based ecosystem to the subject under automatic control.
How to include. In a cloud-based ecosystem under automated control, the subject has the highest or previous probability of having multiple different types of viral antigens and / or antibodies and the communicated data output corresponding to the subject. If you have a virus infection that you are most likely to have, diagnosing the type of virus infection is
Analyzing the communicated data output for positive and / or negative indications for COVID-19 antigen and / or antibody under automatic control.
Under automatic control, positive and / or positive of multiple viral infection microarrays that share at least some of the COVID-19 antigen and / or antibody with the communicated data output for COVID-19 positive and / or negative indications. Comparing the negative indication with the communicated data output,
Under automatic control, the communicated data output of positive and / or negative indications statistically COVID-19 rather than the multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. It is to judge whether or not it is shown in the above, thereby significantly reducing false positives and / or false negatives.
The automatic method according to claim 1. Under automatic control, the communicated data output of positive and / or negative indications statistically COVID-19 rather than the multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. To determine whether or not to indicate, under automatic control, the corresponding Z-score of the communicated data output of the positive and / or negative indication is at least some of the COVID-19 antigen and / or antibody. The automated method of claim 2, comprising determining whether to show COVID-19 rather than the Z-score of the plurality of virus infections to be shared. Under automatic control, the positive and / or positive of the microarray of multiple viral infections that share at least some of the COVID-19 antigen and / or antibody with the communicated data output for COVID-19 positive and / or negative indications. Or to compare with the communicated data output for a negative indicator, under automatic control, the communicated data output for a positive and / or negative indicator of COVID-19, SARS-CoV-2, SARS-CoV, MERS. -Select from a group containing multiple subtypes of CoV, common cold coronavirus (HKU1, OC43, NL63, 229E), and influenza, adenovirus, metapneumovirus, parainfluenza, and / or respiratory conjugation virus. The automated method of claim 2, comprising comparing the positive and / or negative indications of said microarray for a plurality of acute respiratory infections to be communicated data output. Under automatic control, the communicated data output of positive and / or negative indications statistically COVID-19 rather than the multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. The determination is to determine whether or not to indicate, thereby significantly reducing false positives and / or false negatives, the determination is to receive the area under the curve (AUC) is measured under automatic control. Human motion characteristics (ROC) curves are used to assess antigens and distinguish output data from antigen-negative to antigen-positive groups across a wide range of assay cutoff values to identify high-performance antigens that diagnose COVID-19. The automatic method according to claim 2, comprising the above. Under automatic control, the communicated data output of positive and / or negative indications statistically COVID-19 rather than the multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. The determination of whether or not to indicate, thereby significantly reducing false positives and / or false negatives, was calculated with a combination of multiple high performance antigens under automatic control. The automated method of claim 2, comprising identifying the optimal sensitivity and specificity for COVID-19 from a combination of the plurality of high performance antigens based on the corresponding Ioden index. The detection means comprises a microarray of antigen and / or antibody fluorescent spots and, under automatic control, quantitative measurement of the virus antigen and / or antibody in the sample using the corresponding detection means in the instrument. The automatic method according to claim 1, wherein detecting the above comprises generating an image file of a color image of the microarray of antigen and / or antibody fluorescent spots under automatic control. The detection means include a functionalized surface acoustic wave detector (SAW), and under automatic control, the corresponding detection means in the device is used to quantify the viral antigen and / or antibody in the sample. Detection of measurements is, under automatic control, captured directly by the functionalized surface acoustic wave detector (SAW) or by the functionalized surface acoustic wave detector (SAW) corresponding to viral antigens and / or antibodies. The automated method of claim 1, comprising generating an RF phase delayed detection signal in response to quantification of a viral antigen and / or antibody indirectly captured by a polymerase chain reaction (PCR) replicating DNA tag. Under automatic control, the device is used according to the properties of the sample, and the sample is placed on the rotatable disk using the corresponding detection means within the device for the viral antigen and / or antibody to be diagnostically tested. The automated method of claim 1, wherein selective processing comprises performing an ELISA blood test check for immunoglobulin G (IgG) antibody and immunoglobulin M (IgM) antibody under automatic control. Under automatic control, the device is used according to the nature of the sample, and the sample is placed on the rotatable disk using the corresponding detection means within the device for the viral antigen and / or antibody to be diagnostically tested. Selective processing involves performing an immunofluorescence assay under automatic control using a conjugated fluorescent label by direct or indirect immunofluorescence, the amount of conjugate of the antibody to said antigen being the source of fluorescence generation. The automatic method according to claim 1, which directly correlates with the amount. Under automatic control, the device is used according to the nature of the sample, and the sample is placed on the rotatable disk using the corresponding detection means within the device for the viral antigen and / or antibody to be diagnostically tested. Selective processing involves measuring the amount of genetic material (DNA or RNA) in the sample using PCR under automatic control and real-time quantitative polymerase by fast DNA amplification using Taq polymerase. The automated method of claim 1, comprising performing a chain reaction (RT-qPCR). Under automatic control, the device is used according to the nature of the sample, and the sample is placed on the rotatable disk using the corresponding detection means within the device for the viral antigen and / or antibody to be diagnostically tested. To process selectively
Under automatic control, a sample having a first antibody to which a DNA tag is attached and a second antibody having attached magnetic nanoparticles (MNP) is captured from the first portion of the sample. Capturing and capturing a sandwich comprising the first antibody, said second antibody, said sample, said MNP, and said DNA tag.
A predetermined amount of DNA sufficient to overcome the detector's minimum mass sensitivity limit with the DNA tag using isothermal amplification by providing an amount reliably detectable by the detector under automatic control. Duplicate up to the tag and
Placing a second portion of the sample on a microarray under automatic control
To selectively probe the second portion of the sample for an antibody corresponding to at least one selected virus using the microarray under automatic control.
Under automatic control, the microarray is read using a fluorescent camera to identify the antibody in the second portion of the sample.
The automatic method according to claim 1. Under automatic control, the microarray can be used to selectively probe the second portion of the sample for the antibody corresponding to the at least one selected virus.
Culturing the second portion of the sample on the microarray for a predetermined amount of time under automatic control.
Placing the fluorescently labeled secondary Ab under automatic control and
Cleaning the microarray under automatic control and
Drying the microarray under automatic control and
Including
Reading the microarray using a fluorescent camera and identifying the antibody in the second portion of the sample can be done.
To detect the Ig isotype in the second portion of the sample by generating a color image of the microarray under automatic control.
Communicating the color images of the microarray to the cloud for analysis and / or data processing under automated control.
12. The automatic method according to claim 12. The microarray is provided with a DNA spot in the receptacle binding domain (RBD) of the peplomer and, under automatic control, the microarray is used to the antibody corresponding to the at least one selected virus of the sample. Selective probing of the second part is
Under automatic control, the microarray provided with DNA spots for the receptor binding domain (RBD) of the peplomer, fluorescently labeled ACE2, and another fluorescently labeled secondary antibody against human IgG to detect the RBD antibody. To perform a neutralizing antibody assay using
Cleaning the microarray under automatic control and
Drying the microarray under automatic control and
Including
Under automatic control, reading the microarray using a fluorescent camera to identify the antibody in the second portion of the sample can be done.
To generate a color image of the microarray with at least two different colors under automatic control, one color is for the RBD antibody present in said second portion of the sample and the second color is ACE2. The second portion of the sample without neutralizing antibody or RBD antibody is detected using ACE2 fluorescence, while the sample with RBD antibody or the amount of neutralizing antibody that interferes with ACE2-RBD binding is present. Larger samples were detected with reduced ACE2 fluorescence, and in the absence of RBD antibody, the amount of ACE2 fluorescence could be quantified for relative neutralizing activity.
Communicating the color images of the microarray to the cloud for analysis and / or data processing under automated control.
12. The automatic method according to claim 12. Selective probing of the second portion of the sample for an antibody corresponding to the at least one selected virus using the microarray under automatic control uses reciprocating motion under automatic control. The second portion of the sample is then micromixed so that the centrifugal acceleration acting on the liquid element first produces and stores air energy, which is then the centrifuge acceleration. An alternating sequence of micromixing and high and low centrifugation acceleration, released by lowering and resulting in reverse liquid flow direction, is applied to the second portion of the sample and cultured / The automated method of claim 12, further comprising maximizing the culture / hybridization efficiency between the antibody macromolecule and the antigen macromolecule during hybridization. An automated cloud-based system that uses an automated portable handheld device to perform field diagnostic tests on samples taken from a subject to determine the presence of viral antigens and / or antibodies.
The sample receiving chamber in the rotatable disk in the device and
The device is used according to the nature of the sample and the sample is automatically processed on the rotatable disk using the corresponding detection means in the portable handheld device for the viral antigen and / or antibody to be diagnostically tested. With the leader to do
A detector that automatically detects the quantitative measurement of the viral antigen and / or antibody in the sample using the corresponding detection means in the portable handheld device.
A data output circuit that automatically generates data output of the detected quantitative measurement of the virus antigen and / or antibody in the sample corresponding to the subject.
A communication circuit that automatically communicates the data output corresponding to the subject to the cloud-based database,
Under automated control, multiple different types of viral antigens and / or antibodies and the communicated data output corresponding to the subject are compared and analyzed to have the highest or previous probability of the subject having. With a cloud-based ecosystem that diagnoses the type of viral infection, if there is a high viral infection, and automatically communicates the results of the comparative analysis from the cloud-based ecosystem to the subject.
Automatic cloud-based system with. Under the automatic control, a plurality of different types of viral antigens and / or antibodies and the communicated data output corresponding to the subject are compared and analyzed to determine the highest or previous probability that the subject has. If there is the highest viral infection, the cloud-based ecosystem diagnosing the type of viral infection will automatically output the communicated data output of the microarray for positive and / or negative indications for the COVID-19 antigen and / or antibody. The positive and / or positive of the microarray of multiple viral infections that share at least some of the COVID-19 antigen and / or antibody with the communicated data output for COVID-19 positive and / or negative indications. Or automatically compare to the communicated data output for a negative indicator, and said said that the communicated data output of a positive and / or negative indicator shares at least some of the COVID-19 antigen and / or antibody. It is an automatic determination of whether or not COVID-19 is statistically indicated rather than multiple virus infections, thereby significantly reducing false positives and / or false negatives. 16. The automated cloud-based system of claim 16, comprising a cloud-based module for doing so. Whether the communicated data output of positive and / or negative indication statistically indicates COVID-19 rather than the multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. The cloud-based module automatically determines that, under automatic control, the corresponding Z-score of the communicated data output of the positive and / or negative indication is at least some of the COVID-19 antigen and / or antibody. 17. The automatic cloud-based system of claim 17, comprising a cloud-based module that automatically determines whether or not it exhibits COVID-19 rather than the Z-score of the plurality of virus infections that share the same. Under automatic control, the positive and / or positive of the microarray of multiple viral infections that share at least some of the COVID-19 antigen and / or antibody with the communicated data output for COVID-19 positive and / or negative indications. Or the cloud-based module that automatically compares the communicated data output for a negative indication with the communicated data output for a positive and / or negative indication of COVID-19, SARS-CoV-2, SARS-CoV, From a group containing MERS-CoV, common cold coronaviruses (HKU1, OC43, NL63, 229E), and multiple subtypes of influenza, adenovirus, metapneumovirus, parainfluenza, and / or respiratory conjugation virus. 17. The automated cloud-based system of claim 17, comprising a cloud-based module that automatically compares the positive and / or negative indications of said microarray of selected acute respiratory infections with the communicated data output. Under automatic control, the communicated data output of positive and / or negative indications statistically COVID-19 rather than the multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. The cloud-based module that automatically determines whether or not it is indicated in the above, wherein false positives and / or false negatives are significantly reduced, and the cloud-based module that automatically determines the area under the curve ( The receiver operating characteristic (ROC) curve from which AUC) is measured is used to automatically assess antigens and distinguish between antigen-negative and antigen-positive output data over a wide range of assay cutoff values, COVID-. 19. The automated cloud-based system of claim 17, comprising a cloud-based module that identifies a high performance antigen that diagnoses 19. Whether the communicated data output of positive and / or negative indications statistically indicates COVID-19 rather than the multiple viral infections that share at least some of the COVID-19 antigen and / or antibody. The cloud-based module that automatically determines, thereby significantly reducing false positives and / or false negatives, the cloud-based module that automatically determines multiple high performances under automatic control. 17. Claim 17, comprising a cloud-based module that automatically identifies the optimal sensitivity and specificity for COVID-19 from the combination of the plurality of high performance antigens based on the corresponding Ioden index calculated for the combination of antigens. Described automatic cloud-based system.

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