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1.
Nature ; 605(7910): 464-469, 2022 05.
Article in English | MEDLINE | ID: covidwho-1852427

ABSTRACT

Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications1-5. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system)6,7 was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation8-11. Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible12-19. Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are three-dimensionally printed, and powered by the free energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (1) the sequential release of 300 aliquots across chained, interconnected chips, (2) a protocol for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies detection in saliva and (3) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ and can form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.


Subject(s)
COVID-19 , Microfluidic Analytical Techniques , Humans , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques/methods , Microfluidics/methods , Polymerase Chain Reaction , SARS-CoV-2/genetics
2.
ACS Appl Mater Interfaces ; 13(33): 38990-39002, 2021 Aug 25.
Article in English | MEDLINE | ID: covidwho-1351922

ABSTRACT

The ongoing COVID-19 pandemic has clearly established how vital rapid, widely accessible diagnostic tests are in controlling infectious diseases and how difficult and slow it is to scale existing technologies. Here, we demonstrate the use of the rapid affinity pair identification via directed selection (RAPIDS) method to discover multiple affinity pairs for SARS-CoV-2 nucleocapsid protein (N-protein), a biomarker of COVID-19, from in vitro libraries in 10 weeks. The pair with the highest biomarker sensitivity was then integrated into a 10 min, vertical-flow cellulose paper test. Notably, the as-identified affinity proteins were compatible with a roll-to-roll printing process for large-scale manufacturing of tests. The test achieved 40 and 80 pM limits of detection in 1× phosphate-buffered saline (mock swab) and saliva matrices spiked with cell-culture-generated SARS-CoV-2 viruses and is also capable of detection of N-protein from characterized clinical swab samples. Hence, this work paves the way toward the mass production of cellulose paper-based assays which can address the shortages faced due to dependence on nitrocellulose and current manufacturing techniques. Further, the results reported herein indicate the promise of RAPIDS and engineered binder proteins for the timely and flexible development of clinically relevant diagnostic tests in response to emerging infectious diseases.


Subject(s)
Antigens, Viral/analysis , COVID-19 Serological Testing/methods , Nucleocapsid Proteins/analysis , SARS-CoV-2/chemistry , Biomarkers/analysis , Biosensing Techniques , COVID-19/prevention & control , Cellulose/chemistry , Enzyme-Linked Immunosorbent Assay/methods , Fluorescent Dyes/chemistry , Humans , Microfluidic Analytical Techniques/methods , Peptide Library , Protein Binding
3.
Anal Bioanal Chem ; 413(7): 1787-1798, 2021 Mar.
Article in English | MEDLINE | ID: covidwho-1336052

ABSTRACT

Rapid and accurate identification of respiratory tract infection pathogens is of utmost importance for clinical diagnosis and treatment, as well as prevention of pathogen transmission. To meet this demand, a microfluidic chip-based PCR-array system, Onestart, was developed. The Onestart system uses a microfluidic chip packaged with all the reagents required, and the waste liquid is also collected and stored on the chip. This ready-to-use system can complete the detection of 21 pathogens in a fully integrated manner, with sample lysis, nucleic acid extraction/purification, and real-time PCR sequentially implemented on the same chip. The entire analysis process is completed within 1.5 h, and the system automatically generates a test report. The lower limit-of-detection (LOD) of the Onestart assay was determined to be 1.0 × 103 copies·mL-1. The inter-batch variation of cycle threshold (Ct) values ranged from 0.08% to 0.69%, and the intra-batch variation ranged from 0.9% to 2.66%. Analytical results of the reference sample mix showed a 100% specificity of the Onestart assay. The analysis of batched clinical samples showed consistency of the Onestart assay with real-time PCR. With its ability to provide rapid, sensitive, and specific detection of respiratory tract infection pathogens, application of the Onestart system will facilitate timely clinical management of respiratory tract infections and effective prevention of pathogen transmission. Onestart, a ready-to-use system, can detect 21 pathogens in a fully integrated manner on a microchip within 1.5 h.


Subject(s)
Automation , Polymerase Chain Reaction/methods , Respiratory Tract Infections/diagnosis , COVID-19 Testing/methods , Diagnosis, Computer-Assisted , Equipment Design , Humans , Lab-On-A-Chip Devices , Limit of Detection , Microfluidic Analytical Techniques/methods , Microfluidics , Pattern Recognition, Automated , Quality Control , RNA, Viral/analysis , Reproducibility of Results , Respiratory Tract Infections/metabolism , Respiratory Tract Infections/virology , SARS-CoV-2 , Sensitivity and Specificity , Viruses
4.
Mikrochim Acta ; 188(8): 261, 2021 07 19.
Article in English | MEDLINE | ID: covidwho-1316284

ABSTRACT

The ongoing global pandemic of SARS-CoV-2 has promoted to develop novel serological testing technologies since they can be effectively complementary to RT-PCR. Here, a new all-fiber Fresnel reflection microfluidic biosensor (FRMB) was constructed through combining all-fiber optical system, microfluidic chip, and multimode fiber bio-probe. The transmission of the incident light and the collection and transmission of Fresnel reflection light are achieved using a single-multi-mode fiber optic coupler (SMFC) without any other optical separation elements. This compact design greatly simplifies the whole system structure and improves light transmission efficiency, which makes it suitable for the label-free, sensitive, and easy-to-use point-of-care testing (POCT) of targets in nanoliter samples. Based on Fresnel reflection mechanism and immunoassay principle, both the SARS-CoV-2 IgM and IgG antibodies against the SARS-CoV-2 spike protein could be sensitively quantified in 7 min using the secondary antibodies-modified multimode fiber bio-probe. The FRMB performs in one-step, is accurate, label-free, and sensitive in situ/on-site detection of SARS-CoV-2 IgM or IgG in serum with simple dilution only. The limits of detection of SARS-CoV-2 IgM and SARS-CoV-2 IgG were 0.82 ng/mL and 0.45 ng/mL, respectively. Based on our proposed theory, the affinity constants of SARS-CoV-2 IgM or IgG antibody and their respective secondary antibodies were also determined. The FRMB can be readily extended as a universal platform for the label-free, rapid, and sensitive in situ/on-site measurement of other biomarkers and the investigation of biomolecular interaction.


Subject(s)
Antibodies, Viral/blood , Biosensing Techniques/methods , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Microfluidic Analytical Techniques/methods , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Humans , Immunoassay/methods , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin M/blood , Immunoglobulin M/immunology , Limit of Detection , SARS-CoV-2/chemistry , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/immunology
5.
Viruses ; 13(5)2021 05 01.
Article in English | MEDLINE | ID: covidwho-1224249

ABSTRACT

To complement RT-qPCR testing for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, many countries have introduced the use of rapid antigen tests. As they generally display lower real-life performances than expected, their correct positioning as frontline screening is still controversial. Despite the lack of data from daily clinical use, third generation microfluidic assays (such as the LumiraDx SARS-CoV-2 Ag test) have recently been suggested to have similar performances to RT-qPCR and have been proposed as alternative diagnostic tools. By analyzing 960 nasopharyngeal swabs from 960 subjects at the emergency department admissions of a tertiary COVID-19 hospital, LumiraDx assay demonstrated a specificity of 97% (95% CI: 96-98), and a sensitivity of 85% (95% CI: 82-89) in comparison with RT-qPCR, which increases to 91% (95% CI: 86-95) for samples with a cycle threshold ≤ 29. Fifty false-negative LumiraDx-results were confirmed by direct quantification of genomic SARS-CoV-2 RNA through droplet-digital PCR (median (IQR) load = 5880 (1657-41,440) copies/mL). Subgenomic N and E RNAs were detected in 52% (n = 26) and 56% (n = 28) of them, respectively, supporting the presence of active viral replication. Overall, the LumiraDx test complies with the minimum performance requirements of the WHO. Yet, the risk of a misrecognition of patients with active COVID-19 persists, and the need for confirmatory RT-qPCR should not be amended.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , SARS-CoV-2/genetics , Aged , Antigens, Viral/analysis , COVID-19/genetics , COVID-19/immunology , Emergency Service, Hospital , Female , Humans , Italy/epidemiology , Male , Microfluidic Analytical Techniques/methods , Middle Aged , Nasopharynx/virology , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Sensitivity and Specificity
6.
Proc Natl Acad Sci U S A ; 118(20)2021 05 18.
Article in English | MEDLINE | ID: covidwho-1216492

ABSTRACT

The urgency for the development of a sensitive, specific, and rapid point-of-care diagnostic test has deepened during the ongoing COVID-19 pandemic. Here, we introduce an ultrasensitive chip-based antigen test with single protein biomarker sensitivity for the differentiated detection of both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A antigens in nasopharyngeal swab samples at diagnostically relevant concentrations. The single-antigen assay is enabled by synthesizing a brightly fluorescent reporter probe, which is incorporated into a bead-based solid-phase extraction assay centered on an antibody sandwich protocol for the capture of target antigens. After optimization of the probe release for detection using ultraviolet light, the full assay is validated with both SARS-CoV-2 and influenza A antigens from clinical nasopharyngeal swab samples (PCR-negative spiked with target antigens). Spectrally multiplexed detection of both targets is implemented by multispot excitation on a multimode interference waveguide platform, and detection at 30 ng/mL with single-antigen sensitivity is reported.


Subject(s)
Antigens, Viral/isolation & purification , Influenza A virus/isolation & purification , Microfluidic Analytical Techniques/methods , Molecular Diagnostic Techniques/methods , SARS-CoV-2/isolation & purification , Antigens, Viral/immunology , Biosensing Techniques , COVID-19/diagnosis , Fluorescence , Humans , Influenza A virus/immunology , Influenza, Human/diagnosis , Lab-On-A-Chip Devices , Limit of Detection , Nasopharynx/virology , Point-of-Care Systems , SARS-CoV-2/immunology
7.
PLoS One ; 16(4): e0243333, 2021.
Article in English | MEDLINE | ID: covidwho-1183614

ABSTRACT

The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.


Subject(s)
COVID-19/diagnosis , Microfluidic Analytical Techniques/methods , SARS-CoV-2/isolation & purification , Specimen Handling/methods , Adult , COVID-19/virology , COVID-19 Testing/methods , DNA Primers , Diagnostic Tests, Routine/methods , Female , Humans , Male , MicroRNAs/genetics , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Sensitivity and Specificity
8.
Clin Chem ; 67(4): 672-683, 2021 03 31.
Article in English | MEDLINE | ID: covidwho-1165392

ABSTRACT

BACKGROUND: Infectious disease outbreaks such as the COVID-19 (coronavirus disease 2019) pandemic call for rapid response and complete screening of the suspected community population to identify potential carriers of pathogens. Central laboratories rely on time-consuming sample collection methods that are rarely available in resource-limited settings. METHODS: We present a highly automated and fully integrated mobile laboratory for fast deployment in response to infectious disease outbreaks. The mobile laboratory was equipped with a 6-axis robot arm for automated oropharyngeal swab specimen collection; virus in the collected specimen was inactivated rapidly using an infrared heating module. Nucleic acid extraction and nested isothermal amplification were performed by a "sample in, answer out" laboratory-on-a-chip system, and the result was automatically reported by the onboard information platform. Each module was evaluated using pseudovirus or clinical samples. RESULTS: The mobile laboratory was stand-alone and self-sustaining and capable of on-site specimen collection, inactivation, analysis, and reporting. The automated sampling robot arm achieved sampling efficiency comparable to manual collection. The collected samples were inactivated in as short as 12 min with efficiency comparable to a water bath without damage to nucleic acid integrity. The limit of detection of the integrated microfluidic nucleic acid analyzer reached 150 copies/mL within 45 min. Clinical evaluation of the onboard microfluidic nucleic acid analyzer demonstrated good consistency with reverse transcription quantitative PCR with a κ coefficient of 0.979. CONCLUSIONS: The mobile laboratory provides a promising solution for fast deployment of medical diagnostic resources at critical junctions of infectious disease outbreaks and facilitates local containment of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) transmission.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Laboratories , Mobile Health Units , Pathology, Molecular/methods , RNA, Viral/analysis , Adult , Automobiles , COVID-19/epidemiology , COVID-19 Nucleic Acid Testing/instrumentation , Female , Humans , Lab-On-A-Chip Devices , Male , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Middle East Respiratory Syndrome Coronavirus/chemistry , Molecular Diagnostic Techniques/instrumentation , Molecular Diagnostic Techniques/methods , Pandemics , Pathology, Molecular/instrumentation , Robotics , SARS-CoV-2/chemistry
9.
Biosens Bioelectron ; 180: 113088, 2021 May 15.
Article in English | MEDLINE | ID: covidwho-1091933

ABSTRACT

Serial measurement of a large panel of protein biomarkers near the bedside could provide a promising pathway to transform the critical care of acutely ill patients. However, attaining the combination of high sensitivity and multiplexity with a short assay turnaround poses a formidable technological challenge. Here, the authors develop a rapid, accurate, and highly multiplexed microfluidic digital immunoassay by incorporating machine learning-based autonomous image analysis. The assay has achieved 12-plexed biomarker detection in sample volume <15 µL at concentrations < 5 pg/mL while only requiring a 5-min assay incubation, allowing for all processes from sampling to result to be completed within 40 min. The assay procedure applies both a spatial-spectral microfluidic encoding scheme and an image data analysis algorithm based on machine learning with a convolutional neural network (CNN) for pre-equilibrated single-molecule protein digital counting. This unique approach remarkably reduces errors facing the high-capacity multiplexing of digital immunoassay at low protein concentrations. Longitudinal data obtained for a panel of 12 serum cytokines in human patients receiving chimeric antigen receptor-T (CAR-T) cell therapy reveals the powerful biomarker profiling capability. The assay could also be deployed for near-real-time immune status monitoring of critically ill COVID-19 patients developing cytokine storm syndrome.


Subject(s)
COVID-19/immunology , Cytokines/analysis , Image Processing, Computer-Assisted/methods , Immunoassay/methods , Machine Learning , Microarray Analysis/methods , Microfluidic Analytical Techniques/methods , SARS-CoV-2 , Cytokine Release Syndrome , Humans , Immunotherapy, Adoptive , Neural Networks, Computer
10.
Biotechniques ; 69(4): 317-325, 2020 10.
Article in English | MEDLINE | ID: covidwho-1067502

ABSTRACT

PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system.


Subject(s)
Polymerase Chain Reaction/methods , Animals , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/instrumentation , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/virology , Equipment Design , Humans , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , Pneumonia, Viral/virology , Point-of-Care Systems , Polymerase Chain Reaction/instrumentation , SARS-CoV-2
11.
Nature ; 582(7811): 277-282, 2020 06.
Article in English | MEDLINE | ID: covidwho-980299

ABSTRACT

The great majority of globally circulating pathogens go undetected, undermining patient care and hindering outbreak preparedness and response. To enable routine surveillance and comprehensive diagnostic applications, there is a need for detection technologies that can scale to test many samples1-3 while simultaneously testing for many pathogens4-6. Here, we develop Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), a platform for scalable, multiplexed pathogen detection. In the CARMEN platform, nanolitre droplets containing CRISPR-based nucleic acid detection reagents7 self-organize in a microwell array8 to pair with droplets of amplified samples, testing each sample against each CRISPR RNA (crRNA) in replicate. The combination of CARMEN and Cas13 detection (CARMEN-Cas13) enables robust testing of more than 4,500 crRNA-target pairs on a single array. Using CARMEN-Cas13, we developed a multiplexed assay that simultaneously differentiates all 169 human-associated viruses with at least 10 published genome sequences and rapidly incorporated an additional crRNA to detect the causative agent of the 2020 COVID-19 pandemic. CARMEN-Cas13 further enables comprehensive subtyping of influenza A strains and multiplexed identification of dozens of HIV drug-resistance mutations. The intrinsic multiplexing and throughput capabilities of CARMEN make it practical to scale, as miniaturization decreases reagent cost per test by more than 300-fold. Scalable, highly multiplexed CRISPR-based nucleic acid detection shifts diagnostic and surveillance efforts from targeted testing of high-priority samples to comprehensive testing of large sample sets, greatly benefiting patients and public health9-11.


Subject(s)
CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems/genetics , Microfluidic Analytical Techniques/methods , Virus Diseases/diagnosis , Virus Diseases/virology , Animals , Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Drug Resistance, Viral/genetics , Genome, Viral/genetics , HIV/classification , HIV/genetics , HIV/isolation & purification , Humans , Influenza A virus/classification , Influenza A virus/genetics , Influenza A virus/isolation & purification , Microfluidic Analytical Techniques/instrumentation , RNA, Guide/genetics , SARS-CoV-2 , Sensitivity and Specificity
12.
Anal Chem ; 92(21): 14297-14302, 2020 11 03.
Article in English | MEDLINE | ID: covidwho-872628

ABSTRACT

With the global outbreak of the coronavirus disease 2019 (COVID-19), the highly infective, highly pathogenic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has attracted great attention. Currently, a method to simultaneously diagnose the seven known types human coronaviruses remains lacking and is urgently needed. In this work, we successfully developed a portable microfluidic system for the rapid, accurate, and simultaneous detection of SARS-CoV, middle east respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2, and four other human coronaviruses (HCoVs) including HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1. The disk-like microfluidic platform integrated with loop-mediated isothermal amplification provides highly accurate, sensitive, and specific results with a wide linear range within 40 min. The diagnostic tool achieved 100% consistency with the "gold standard" polymerase chain reaction in detecting 54 real clinical samples. The integrated system, with its simplicity, is urgently needed for the diagnosis of SARS-CoV-2 during the COVID-19 pandemic.


Subject(s)
Coronavirus Infections/diagnostic imaging , DNA, Viral/analysis , Lab-On-A-Chip Devices , Microfluidic Analytical Techniques/methods , Pneumonia, Viral/diagnostic imaging , Betacoronavirus , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques , Coronavirus 229E, Human , Coronavirus Infections/diagnosis , Coronavirus NL63, Human , Diagnosis, Differential , Humans , Microfluidic Analytical Techniques/instrumentation , Nucleic Acid Amplification Techniques/methods , Pandemics , SARS-CoV-2
14.
Anal Chem ; 92(14): 9454-9458, 2020 07 21.
Article in English | MEDLINE | ID: covidwho-626171

ABSTRACT

The outbreak of SARS-CoV-2 is posing serious global public health problems. Facing the emergence of this pandemic, we established a portable microfluidic immunoassay system for easy-to-use, sensitive, rapid (<15 min), multiple, and on-site detection of IgG/IgM/Antigen of SARS-CoV-2 simultaneously. This integrated method was successfully applied for detecting SARS-CoV-2 IgM and IgG antibodies in clinical human serum as well as SARS-CoV-2 antigen in pharyngeal swabs from 26 patients with COVID-19 infection and 28 uninfected people. The assay demonstrated high sensitivity and specificity, which is promising for the diagnosis and monitoring as well as control of SARS-CoV-2 worldwide.


Subject(s)
Antibodies, Viral/analysis , Betacoronavirus/immunology , Coronavirus Infections/diagnosis , Immunoassay/methods , Immunoglobulin G/analysis , Immunoglobulin M/analysis , Microfluidic Analytical Techniques/methods , Pneumonia, Viral/diagnosis , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/methods , Enzyme-Linked Immunosorbent Assay , Humans , Pandemics , Pharynx/chemistry , Pharynx/immunology , SARS-CoV-2 , Sensitivity and Specificity
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