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1.
Biosensors (Basel) ; 12(10)2022 Sep 23.
Article in English | MEDLINE | ID: covidwho-2043580

ABSTRACT

The global pandemic of COVID-19 has created an unrivalled need for sensitive and rapid point-of-care testing (POCT) methods for the detection of infectious viruses. For the novel coronavirus SARS-CoV-2, the nucleocapsid protein (N-protein) is one of the most abundant structural proteins of the virus and it serves as a useful diagnostic marker for detection. Herein, we report a fiber optic particle plasmon resonance (FOPPR) biosensor which employed a single-stranded DNA (ssDNA) aptamer as the recognition element to detect the SARS-CoV-2 N-protein in 15 min with a limit of detection (LOD) of 2.8 nM, meeting the acceptable LOD of 106 copies/mL set by the WHO target product profile. The sensor chip is a microfluidic chip based on the balance between the gravitational potential and the capillary force to control fluid loading, thus enabling the power-free auto-flowing function. It also has a risk-free self-contained design to avoid the risk of the virus leaking into the environment. These findings demonstrate the potential for designing a low-cost and robust POCT device towards rapid antigen detection for early screening of SARS-CoV-2 and its related mutants.


Subject(s)
Biosensing Techniques , COVID-19 , Humans , SARS-CoV-2 , DNA, Single-Stranded , Microfluidics , COVID-19/diagnosis , Nucleocapsid Proteins/genetics
2.
Anal Bioanal Chem ; 414(28): 7957-7965, 2022 Nov.
Article in English | MEDLINE | ID: covidwho-2035028

ABSTRACT

SARS-CoV-2 has mutated many times since the onset of the COVID-19 pandemic, and the omicron is currently the most dominant variant. Determining the specific strain of the virus is beneficial in providing proper care and containment of the disease. We have previously reported a novel method of counting the number of particle immunoagglutination on a paper microfluidic chip using a smartphone-based fluorescence microscope. A single-copy-level detection was demonstrated from clinical saline gargle samples. In this work, we further evaluated two different SARS-CoV-2 monoclonal antibodies to spike vs. nucleocapsid antigens for detecting omicron vs. delta and spike vs. nucleocapsid proteins. The SARS-CoV-2 monoclonal antibody to nucleocapsid proteins could distinguish omicron from delta variants and nucleocapsid from spike proteins. However, such distinction could not be found with the monoclonal antibody to spike proteins, despite the numerous mutations found in spike proteins among variants. This result may suggest a clue to the role of nucleocapsid proteins in recognizing different variants.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , COVID-19/diagnosis , Spike Glycoprotein, Coronavirus , Pandemics , Microfluidics , Antibodies, Viral , Nucleocapsid Proteins/genetics , Immunoassay , Antibodies, Monoclonal
3.
Front Bioeng Biotechnol ; 10: 895236, 2022.
Article in English | MEDLINE | ID: covidwho-1952241

ABSTRACT

Ultrafast, portable, and inexpensive molecular diagnostic platforms are critical for clinical diagnosis and on-site detection. There are currently no available real-time polymerase chain reaction (PCR) devices able to meet the demands of point-of-care testing, as the heating and cooling processes cannot be avoided. In this study, the dual temperature modules were first designed to process microfluidic chips automatically circulating between them. Thus, a novel ultrafast molecular diagnostic real-time PCR device (approximately 18 and 23 min for DNA and RNA detection, respectively) with two channels (FAM and Cy5) for the detection of 12 targets was developed. The device contained three core functional components, including temperature control, optics, and motion, which were integrated into a portable compact box. The temperature modules accurately control temperature in rapid thermal cycles with less than ±0.1 °C, ±1 °C and ±0.5 °C for the temperature fluctuation, uniformity, and error of indication, respectively. The average coefficient of variation (CV) of the fluorescence intensity (FI) for all 12 wells was 2.3% for FAM and 2.7% for Cy5. There was a good linear relationship between the concentrations of fluorescent dye and the FIs of FAM and Cy5(R 2 = 0.9990 and 0.9937), and the average CVs of the Ct values calculated by the embedded software were 1.4% for FAM and Cy5, respectively. The 100 double-blind mocked sputum and 249 clinical stool samples were analyzed by the ultrafast real-time PCR device in comparison with the DAAN Gene SARS-CoV-2 kit run on the ABI 7500 instrument and Xpert C. difficile/Epi, respectively. Among the 249 stool samples, the ultrafast real-time PCR device detected toxigenic C. difficile in 54 samples (54/249, 21.7%) with a specificity and positive predictive values of 99.0 and 96.3%, which were higher than the Xpert C. difficile/Epi values of 94.4 and 88.1% (p > 0.05). The ultrafast real-time PCR device detected 15 SARS-CoV-2 positive samples, which has a 100% concordance with that obtained by the DAAN Gene SARS-CoV-2 kit. This study demonstrated that the ultrafast real-time PCR device integrated with microfluidic chips and dual temperature modules is an ultrafast, reliable, easy-to-use, and cost-effective molecular diagnostic platform for clinical diagnosis and on-site testing, especially in resource-limited settings.

4.
Biosens Bioelectron ; 210: 114293, 2022 Aug 15.
Article in English | MEDLINE | ID: covidwho-1797125

ABSTRACT

In the wake of a pandemic, the development of rapid, simple, and accurate molecular diagnostic tests can significantly aid in reducing the spread of infections. By combining particle imaging with molecular assays, a quick and highly sensitive biosensor can readily identify a pathogen at low concentrations. Here, we implement functionalized particle-enabled rotational diffusometry in combination with loop-mediated isothermal amplification for the rapid detection of the SARS-CoV-2 nsp2 gene in the recombinant plasmid as a proof of concept for COVID-19 diagnostics. By analyzing the images of blinking signals generated by these modified particles, the change in micro-level viscosity due to nucleic acid amplification was measured. The high sensitivity of rotational diffusometry enabled facile detection within 10 min, with a limit of detection of 70 ag/µL and a sample volume of 2 µL. Tenfold higher detection sensitivity was observed for rotational diffusometry in comparison with real-time PCR. In addition, the system stability and the effect of temperature on rotational diffusometric measurements were studied and reported. These results demonstrated the utility of a rotational diffusometric platform for the rapid and sensitive detection of SARS-CoV-2 cDNA fragments.


Subject(s)
Biosensing Techniques , COVID-19 , COVID-19/diagnosis , DNA, Complementary , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , RNA, Viral/genetics , SARS-CoV-2/genetics , Sensitivity and Specificity
5.
Sheng Wu Gong Cheng Xue Bao ; 38(3): 943-960, 2022 Mar 25.
Article in Chinese | MEDLINE | ID: covidwho-1771822

ABSTRACT

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics. The PCR includes multiple reaction stages (denaturation, annealing, and extension), and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30-40 cycles within at least 1-2 hours. Due to the complicated devices and the long amplification time, it is difficult to adopt conventional PCR in point-of-care testing (POCT). Comparing to conventional PCR, isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device. When isothermal amplification is combined with microfluidics, a more competent platform for POCT can be established. For example, various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses. This review summarized the recent development and applications of the microfluidics-based isothermal amplification. First, different typical isothermal amplification methods and related detection methods have been introduced. Subsequently, different types of microfluidic systems with isothermal amplification were discussed based on their characteristics, for example, functionality, system structure, flow control, and operation principles. Furthermore, detection of pathogens (e.g. SARS-CoV-2 viruses) based on isothermal amplification was introduced. Finally, the combination of isothermal amplification with other new technologies, e.g. CRISPR, has been introduced as well.


Subject(s)
COVID-19 , Microfluidics , COVID-19/diagnosis , Humans , Nucleic Acid Amplification Techniques , Polymerase Chain Reaction , SARS-CoV-2/genetics
6.
Chinese Journal of Analytical Chemistry ; 50(1):25-38, 2022.
Article in Chinese | Web of Science | ID: covidwho-1667872

ABSTRACT

Digital polymerase chain reaction (PCR), as a nucleic acid detection technology with wide application prospect, has become one of the most accurate nucleic acid detection technology at present. Multiplex detection is an important direction for the development of digital PCR technique. With the development of microfluidic technology, multiplex digital PCR technique has become more and more mature. This paper reviewed the research progresses of multiplex digital PCR in recent years, especially summarized the implementation of multiplex digital PCR technique in the past five years, and introduced the application of multiplex digital PCR technique in hot areas such as liquid biopsy, transgenic detection, and SARS-Cov-2 detection. Finally, the issues and challenges faced by multiplex digital PCR technique were discussed and the future direction of the technology was foreseen.

7.
Micromachines (Basel) ; 13(2)2022 Jan 28.
Article in English | MEDLINE | ID: covidwho-1667244

ABSTRACT

In the context of the COVID-19 epidemic, enhancing the transport of analyte to a sensor surface is crucial for rapid detection of biomolecules since common conditions, including low diffusion coefficients, cause inordinately long detection times. Integrated microfluidic immunoassay chips are receiving increasing attention for their low sample volume and fast response time. We herein take advantage of asymmetric ICEO flow at a bipolar sinusoidal electrode to improve the rate of antibody binding to the reaction surface based on finite element modeling. Three different microfluidic cavities are proposed by changing the positions of the surface reaction area. We further investigate the relationship between binding enhancement and reaction surface positions, Damkohler number, and the voltage and frequency of the AC signal applied to the driving electrodes. Furthermore, the influence of the AC signal applied to the sinusoidal bipolar electrode on antigen-antibody-binding performance is studied in detail. Above all, the simulation results demonstrate that the microfluidic immune-sensor with a sinusoidal bipolar electrode could not only significantly improve the heterogeneous immunoassays but also enable efficient enhancement of assays in a selected reaction region within the micro-cavity, providing a promising approach to a variety of immunoassay applications, such as medical diagnostics and environmental and food monitoring.

8.
Optics in Health Care and Biomedical Optics XI 2021 ; 11900, 2021.
Article in English | Scopus | ID: covidwho-1621985

ABSTRACT

Acquisition of the genes encoding variable regions of paired heavy and light chains (VH:VL) is crucial, but it is a labor and cost-intensive process in traditional methods. This study presents a novel method in which all processing steps for acquiring natively paired VH:VL genes from single cells are finished in a single microfluidic chip. The microfluidic chip performs single-cell trap/in situ fluorescent examination of antibody specificity/cell lysis/gene amplification all at single-cell level. By a proof-of-concept validation of efficiently acquiring paired VH:VL genes of anti-RBD (which is a key protein of SARS-CoV-2 virus) mAbs from single hybridomas, the microfluidic chip has been proved capable of remarkably improving cell loss/human labor/time cost, and more importantly, determinacy of native VH:VL genes pairing which is one of the most decisive factors of effectiveness for antibody discovery. © 2021 SPIE.

9.
Micromachines (Basel) ; 12(12)2021 Dec 19.
Article in English | MEDLINE | ID: covidwho-1580576

ABSTRACT

A two-stage isothermal amplification method, which consists of a first-stage basic recombinase polymerase amplification (RPA) and a second-stage fluorescence loop-mediated isothermal amplification (LAMP), as well as a microfluidic-chip-based portable system, were developed in this study; these enabled parallel detection of multiplex targets in real time in around one hour, with high sensitivity and specificity, without cross-contamination. The consumption of the sample and the reagent was 2.1 µL and 10.6 µL per reaction for RPA and LAMP, respectively. The lowest detection limit (LOD) was about 10 copies. The clinical amplification of about 40 nasopharyngeal swab samples, containing 17 SARS-CoV-2 (severe acute respiratory syndrome coronavirus) and 23 measles viruses (MV), were parallel tested by using the microfluidic chip. Both clinical specificity and sensitivity were 100% for MV, and the clinical specificity and sensitivity were 94.12% and 95.83% for SARS-CoV-2, respectively. This two-stage isothermal amplification method based on the microfluidic chip format offers a convenient, clinically parallel molecular diagnostic method, which can identify different nucleic acid samples simultaneously and in a timely manner, and with a low cost of the reaction reagent. It is especially suitable for resource-limited areas and point-of-care testing (POCT).

10.
Bioengineered ; 13(1): 876-883, 2022 01.
Article in English | MEDLINE | ID: covidwho-1585254

ABSTRACT

This research has developed a method for rapid detection of SARS-CoV-2 N protein on a paper-based microfluidic chip. The chitosan-glutaraldehyde cross-linking method is used to fix the coated antibody, and the sandwich enzyme-linked immunosorbent method is used to achieve the specific detection of the target antigen. The system studied the influence of coating antibody concentration and enzyme-labeled antibody concentration on target antigen detection. According to the average gray value measured under different N protein concentrations, the standard curve of the method was established and the sensitivity was tested, and its linear regression was obtained. The equation is y = 9.8286x+137.6, R2 = 0.9772 > 0.90, which shows a high degree of fit. When the concentration of coating antibody and enzyme-labeled antibody were 1 µg/mL and 2 µg/mL, P > 0.05, the difference was not statistically significant, so the lower concentration of 1 µg/mL was chosen as the coating antibody concentration. The results show that the minimum concentration of N protein that can be detected by this method is 8 µg/mL, and the minimum concentration of coating antibody and enzyme-labeled antibody is 1 µg/mL, which has the characteristics of high sensitivity and good repeatability.


Subject(s)
Antigens, Viral/analysis , COVID-19 Serological Testing/instrumentation , Coronavirus Nucleocapsid Proteins/analysis , Coronavirus Nucleocapsid Proteins/immunology , Lab-On-A-Chip Devices , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Antibodies, Viral/immunology , Biomedical Engineering , COVID-19/diagnosis , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/methods , COVID-19 Serological Testing/standards , Coronavirus Nucleocapsid Proteins/standards , Enzyme-Linked Immunosorbent Assay/instrumentation , Enzyme-Linked Immunosorbent Assay/methods , Enzyme-Linked Immunosorbent Assay/standards , Humans , Lab-On-A-Chip Devices/standards , Lab-On-A-Chip Devices/statistics & numerical data , Microchip Analytical Procedures/methods , Microchip Analytical Procedures/standards , Microchip Analytical Procedures/statistics & numerical data , Paper , Phosphoproteins/analysis , Phosphoproteins/immunology , Phosphoproteins/standards
11.
Biosens Bioelectron ; 199: 113865, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1560782

ABSTRACT

Rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for early diagnostics and timely medical treatment of coronavirus disease 2019 (COVID-19). However, current detection methods typically rely on expensive and bulky instrumentation. Here, we developed a simple, sensitive, instrument-free, CRISPR-based diagnostics of SARS-CoV-2 using a self-contained microfluidic system. The microfluidic chip integrates isothermal amplification, CRISPR cleavage, and lateral flow detection in a single, closed microfluidic platform, enabling contamination-free, visual detection. To simplify the operation and transportation of the device, we lyophilized the CRISPR reagents in the reaction chamber and pre-stored the liquid solutions in blisters. We employed a low-cost, portable hand warmer to incubate the microfluidic chip without the need for electricity. The self-contained microfluidic system can detect down to 100 copies of SARS-CoV-2 RNA. Further, we clinically validated our method by detecting 24 COVID-19 clinical nasopharyngeal swab samples, achieving excellent sensitivity (94.1%), specificity (100%), and accuracy (95.8%). This simple, sensitive, and affordable microfluidic system represents a promising tool for point-of-care diagnostics of COVID-19 and other infectious diseases.


Subject(s)
Biosensing Techniques , COVID-19 , CRISPR-Cas Systems , Humans , Microfluidics , Nucleic Acid Amplification Techniques , RNA, Viral/genetics , SARS-CoV-2 , Sensitivity and Specificity
12.
Adv Sci (Weinh) ; 8(23): e2103266, 2021 12.
Article in English | MEDLINE | ID: covidwho-1479368

ABSTRACT

Activation of endothelial cells following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is thought to be the primary driver for the increasingly recognized thrombotic complications in coronavirus disease 2019 patients, potentially due to the SARS-CoV-2 Spike protein binding to the human angiotensin-converting enzyme 2 (hACE2). Vaccination therapies use the same Spike sequence or protein to boost host immune response as a protective mechanism against SARS-CoV-2 infection. As a result, cases of thrombotic events are reported following vaccination. Although vaccines are generally considered safe, due to genetic heterogeneity, age, or the presence of comorbidities in the population worldwide, the prediction of severe adverse outcome in patients remains a challenge. To elucidate Spike proteins underlying patient-specific-vascular thrombosis, the human microcirculation environment is recapitulated using a novel microfluidic platform coated with human endothelial cells and exposed to patient specific whole blood. Here, the blood coagulation effect is tested after exposure to Spike protein in nanoparticles and Spike variant D614G in viral vectors and the results are corroborated using live SARS-CoV-2. Of note, two potential strategies are also examined to reduce blood clot formation, by using nanoliposome-hACE2 and anti-Interleukin (IL) 6 antibodies.


Subject(s)
Blood Coagulation/physiology , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Antibodies/chemistry , Antibodies/immunology , Antibodies/metabolism , COVID-19/diagnosis , COVID-19/virology , Endothelial Cells/chemistry , Endothelial Cells/cytology , Endothelial Cells/metabolism , Fibrin/chemistry , Fibrin/metabolism , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Interleukin-6/immunology , Liposomes/chemistry , Microfluidics/methods , Mutation , Nanoparticles/chemistry , Platelet Aggregation , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/analysis , Spike Glycoprotein, Coronavirus/genetics
13.
Talanta ; 239: 122974, 2022 Mar 01.
Article in English | MEDLINE | ID: covidwho-1466916

ABSTRACT

Direct RNA detection is critical for providing the RNA insights into gene expression profiling, noncoding RNAs, RNA-associated diseases and pathogens, without reverse transcription. However, classical RNA analysis usually requires RT-PCR, which can cause bias amplification and quantitation errors. To address this challenge, herein we report a microfluidic RNA chip (the microchip prototype) for direct RNA detection, which is primarily based on RNA extension and labeling with DNA polymerase. This detection strategy is of high specificity (discriminating against single-nucleotide differences), rapidity, accuracy, nuclease resistance, and reusability. Further, we have successfully detected disease-associated RNAs in clinical samples, demonstrating its great potentials in biomedical research and clinical diagnosis.


Subject(s)
Microfluidic Analytical Techniques , RNA , Microfluidics , Nucleotides , Oligonucleotide Array Sequence Analysis , RNA/genetics
14.
Talanta ; 236: 122847, 2022 Jan 01.
Article in English | MEDLINE | ID: covidwho-1401881

ABSTRACT

Nucleocapsid protein (N protein) is the most abundant protein in SARS-CoV2 and is highly conserved, and there are no homologous proteins in the human body, making it an ideal biomarker for the early diagnosis of SARS-CoV2. However, early detection of clinical specimens for SARS-CoV2 remains a challenge due to false-negative results with viral RNA and host antibodies based testing. In this manuscript, a microfluidic chip with femtoliter-sized wells was fabricated for the sensitive digital detection of N protein. Briefly, ß-galactosidase (ß-Gal)-linked antibody/N protein/aptamer immunocomplexes were formed on magnetic beads (MBs). Afterwards, the MBs and ß-Gal substrate fluorescein-di-ß-d-galactopyranoside (FDG) were injected into the chip together. Each well of the chip would only hold one MB as confined by the diameter of the wells. The MBs in the wells were sealed by fluorocarbon oil, which confines the fluorescent (FL) product generated from the reaction between ß-Gal and FDG in the individual femtoliter-sized well and creates a locally high concentration of the FL product. The FL images of the wells were acquired using a conventional inverted FL microscope. The number of FL wells with MBs (FL wells number) and the number of wells with MBs (MBs wells number) were counted, respectively. The percentage of FL wells was calculated by dividing (FL wells number) by (MBs wells number). The higher the percentage of FL wells, the higher the N protein concentration. The detection limit of this digital method for N protein was 33.28 pg/mL, which was 300 times lower than traditional double-antibody sandwich based enzyme-linked immunosorbent assay (ELISA).


Subject(s)
Immunoassay/methods , Nucleocapsid Proteins , SARS-CoV-2 , Antibodies , COVID-19/diagnosis , Humans , Nucleocapsid Proteins/isolation & purification , RNA, Viral
15.
Int J Pharm ; 607: 121032, 2021 Sep 25.
Article in English | MEDLINE | ID: covidwho-1364122

ABSTRACT

Nanotechnology has provided novel approaches against food born and pathogenic bacteria. Within the present study, the effects of pure and nanoemulsified essential oil derived from Satureja Khuzistanica essential oil (SKEO) on Escherichia coli (E. coli ATCC 25922) as a human pathogen has been studied using a microfluidic chip. The morphology and antibacterial activity of E. coli at disparate residence durations (from 2 to 30 min) and various nanoemulsified or pure essential oil concentrations (8.0-62.5 µg mL-1) and numerous nanoemulsion's droplet sizes from 32 to 124 nm, have been investigated in the microfluidic system. Also, the quantitative analysis including optical density, time killing assay, protein, nucleic acid and potassium release were employed to confirm the effects of bacterial inhibition taking advantage of the chip apparatus. It was revealed that the prepared nanoemulsion left a considerable destructive effect on E. coli bacterial membrane, confirmed by fast release of cytoplasmic elements including protein, nucleic acid and potassium. However, this process was remarkably intensified for both nanoemulsion and pure essential oil using the microfluidic chip versus the conventional methods. The results also revealed that after 4 min of bacterium treatment by 12.5 µg mL-1 nanoemulsion with 32 nm mean particle size, the bacterial membrane wall began to degrade rapidly, and bacterial activity was almost completely inhibited in a 20-min period. These findings may have implications in the similarly structured and phospholipid-encapsulated bacteria and viruses, like COVID-19.


Subject(s)
COVID-19 , Oils, Volatile , Satureja , Escherichia coli , Humans , Microfluidics , Oils, Volatile/pharmacology , SARS-CoV-2
16.
Micromachines (Basel) ; 12(7)2021 Jul 05.
Article in English | MEDLINE | ID: covidwho-1323305

ABSTRACT

As an important route for disease transmission, bioaerosols have received increasing attention. In the past decades, many efforts were made to facilitate the development of bioaerosol monitoring; however, there are still some important challenges in bioaerosol collection and detection. Thus, recent advances in bioaerosol collection (such as sedimentation, filtration, centrifugation, impaction, impingement, and microfluidics) and detection methods (such as culture, molecular biological assay, and immunological assay) were summarized in this review. Besides, the important challenges and perspectives for bioaerosol biosensing were also discussed.

17.
Biosens Bioelectron ; 192: 113499, 2021 Nov 15.
Article in English | MEDLINE | ID: covidwho-1309166

ABSTRACT

The recent outbreak of COVID-19 has highlighted the seriousness of airborne diseases and the need for a proper pathogen detection system. Compared to the ample amount of research on biological detection, work on integrated devices for air monitoring is rare. In this work, we integrated a wet-cyclone air sampler and a DC impedance microfluidic cytometer to build a cyclone-cytometer integrated air monitor (CCAM). The wet-cyclone air sampler sucks the air and concentrates the bioaerosols into 10 mL of aqueous solvent. After 5 min of air sampling, the bioaerosol-containing solution was conveyed to the microfluidic cytometer for detection. The device was tested with aerosolized microbeads, dust, and Escherichia coli (E. coli). CCAM is shown to differentiate particles from 0.96 to 2.95 µm with high accuracy. The wet cyclone air-sampler showed a 28.04% sampling efficiency, and the DC impedance cytometer showed 87.68% detection efficiency, giving a total of 24.59% overall CCAM efficiency. After validation of the device performance, CCAM was used to detect bacterial aerosols and their viability without any separate pretreatment step. Differentiation of dust, live E. coli, and dead E. coli was successfully performed by the addition of BacLight bacterial viability reagent in the sampling solvent. The usage could be further extended to detection of specific species with proper antibody fluorescent label. A promising strategy for aerosol detection is proposed through the constructive integration of a DC impedance microfluidic cytometer and a wet-cyclone air sampler.


Subject(s)
Biosensing Techniques , COVID-19 , Cyclonic Storms , Aerosols/analysis , Air Microbiology , Electric Impedance , Environmental Monitoring , Escherichia coli , Humans , Microfluidics , SARS-CoV-2
18.
Sens Actuators B Chem ; 344: 130242, 2021 Oct 01.
Article in English | MEDLINE | ID: covidwho-1260865

ABSTRACT

Severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic has become a global public health emergency. The detection of SARS-CoV-2 and human enteric pathogens in wastewater can provide an early warning of disease outbreak. Herein, a sensitive, multiplexed, colorimetric detection (termed "SMCD") method was established for pathogen detection in wastewater samples. The SMCD method integrated on-chip nucleic acid extraction, two-stage isothermal amplification, and colorimetric detection on a 3D printed microfluidic chip. The colorimetric signal during nucleic acid amplification was recorded in real-time and analyzed by a programmed smartphone without the need for complicated equipment. By combining two-stage isothermal amplification assay into the integrated microfluidic platform, we detected SARS-CoV-2 and human enteric pathogens with sensitivities of 100 genome equivalent (GE)/mL and 500 colony-forming units (CFU)/mL, respectively, in wastewater within one hour. Additionally, we realized smart, connected, on-site detection with a reporting framework embedded in a portable detection platform, which exhibited potential for rapid spatiotemporal epidemiologic data collection regarding the environmental dynamics, transmission, and persistence of infectious diseases.

19.
Biosens Bioelectron ; 176: 112920, 2021 Mar 15.
Article in English | MEDLINE | ID: covidwho-1002363

ABSTRACT

The worldwide epidemic of novel coronavirus disease (COVID-19) has led to a strong demand for highly efficient immunobinding to achieve rapid and accurate on-site detection of SARS-CoV-2 antibodies. However, hour-scale time-consumption is usually required to ensure the adequacy of immunobinding on expensive large instruments in hospitals, and the common false negative or positive results often occur in rapid on-site immunoassay (e.g. immunochromatography). We solved this dilemma by presenting a reciprocating-flowing immunobinding (RF-immunobinding) strategy. RF-immunobinding enabled the antibodies in fluid contacting with the corresponding immobilized antigens on substrate repeatedly during continuous reciprocating-flowing, to achieve adequate immunobinding within 60 s. This strategy was further developed into an immunoassay method for the serological detection of 13 suspected COVID-19 patients. We obtained a 100% true negative and true positive rate and a limit of quantification (LOQ) of 4.14 pg/mL. Our strategy also can be a potential support for other areas related to immunorecognition, such as proteomics, immunopharmacology and immunohistochemistry.


Subject(s)
COVID-19 Serological Testing/instrumentation , COVID-19/diagnosis , Lab-On-A-Chip Devices , SARS-CoV-2/immunology , Antibodies, Viral/blood , Antigen-Antibody Reactions , Biosensing Techniques/instrumentation , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing/methods , Enzyme-Linked Immunosorbent Assay/instrumentation , Equipment Design , Humans , Immobilized Proteins , Pandemics
20.
Neurobiol Dis ; 146: 105131, 2020 12.
Article in English | MEDLINE | ID: covidwho-872391

ABSTRACT

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system include neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-CoV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. The spike protein, which plays a key role in receptor recognition, is formed by the S1 subunit containing a receptor binding domain (RBD) and the S2 subunit. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is ubiquitously expressed throughout various vessel calibers in the frontal cortex. Moreover, ACE2 expression was upregulated in cases of hypertension and dementia. ACE2 was also detectable in primary hBMVECs maintained under cell culture conditions. Analysis of cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48 h exposure window. Introduction of spike proteins to invitro models of the blood-brain barrier (BBB) showed significant changes to barrier properties. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluidic model of the human BBB, a platform that more closely resembles the physiological conditions at this CNS interface. Evidence provided suggests that the SARS-CoV-2 spike proteins trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Blood-Brain Barrier/metabolism , Capillary Permeability/physiology , Endothelial Cells/metabolism , Inflammation/metabolism , Matrix Metalloproteinases/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/physiology , Blood-Brain Barrier/drug effects , COVID-19 , Capillary Permeability/drug effects , Cell Adhesion Molecules/drug effects , Cell Adhesion Molecules/metabolism , Cell Survival/drug effects , Dementia/metabolism , Electric Impedance , Endothelial Cells/drug effects , Frontal Lobe/metabolism , Humans , Hypertension/metabolism , In Vitro Techniques , Intercellular Junctions/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Lab-On-A-Chip Devices , Matrix Metalloproteinases/drug effects , Primary Cell Culture , Protein Domains , Protein Subunits/metabolism , Protein Subunits/pharmacology , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Spike Glycoprotein, Coronavirus/pharmacology
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