Highly sensitive digital detection of SARS-CoV-2 nucleocapsid protein through single-molecule counting.

Nucleocapsid protein (NP) is one of the structural proteins of SARS-CoV-2 which is stable, well-conserved, highly immunogenic, and abundantly expressed due to the host's adaptive immune response, making it a promising antigenic biomarker for the early and rapid identification and diagnosis of SARS-CoV-2. Traditional antigen analytical methods with NP as the detection marker often have insufficient sensitivity. To achieve rapid and highly sensitive detection of NP, we constructed a novel single-molecule (digital) fluorescence-linked immunosorbent assay (FLISA) based on streptavidin-modified transparent 96-well microplates. Streptavidin was immobilized on the microplate under optimized conditions with a 15 mM carbonate buffer solution (pH 9.6) as the coating solution, biotinylated antibodies conjugated with streptavidin as capture probes, and carboxylated fluorescent microsphere-conjugated monoclonal antibodies (FMs-mAbs) as fluorescent probes. Individual sandwich immunolabeled complexes of the SARS-CoV-2 diagnostic marker NP were detected and counted though wide-field inverted fluorescence microscopy (1.1 × 1.4 mm2). FLISA had a linear detection range of 0.2 pg/mL to 200 ng/mL and a limit of detection (LOD) of 0.73 fg/mL and 8 fg/mL for NP in phosphate buffer saline and spiked nasal swab samples, respectively. The sensitivity was much higher than commercial antigen detection kits, providing wide detection prospects in future clinical diagnosis, environmental monitoring, and other fields.

Quantitative analysis of respiratory viruses based on lab-on-a-chip platform.

The quantitative analysis of respiratory viruses is of great importance for rapid diagnosis, precision medicine, and prognosis. Several current quantitative analysis systems have been proposed and commercialized. Although they have been proven in trials, quantitative analyzes based on real samples are still complex, time-consuming, and expensive. Therefore, they are not able to directly quantify real samples. In this work, we presented a lab-on-a-chip platform combined with an automated control system to achieve quantitative analysis from samples to results. We developed a multilayer integrated chip to rapidly extract and quantify RNA of coronavirus disease 2019 (COVID-19) pseudovirus from large-volume nasal swab samples. The dependence of the magnetic bead size and the interfacial effect was studied for the first time, and the conditions of immiscible filtration assisted by surface tension (IFAST) method for nucleic acid extraction were optimized to increase the nucleic acid recovery rate up to 85%. Inside the chip, a pneumatic valve was developed for automatic opening and closing of the liquid channel. The integrated chip platform and automatic control system presented here are advantageous for use in resource-limited settings (RLS). In addition, our method can be extended to other respiratory viruses and other sample types.

Fabrication of planar monolayer microreactor array for visual statistical analysis and droplet-based digital quantitative analysis in situ.

Planar monolayer microreactor arrays (PMMRAs) make droplet-based numerical measurements and statistical analysis cheap and easy. However, PMMRAs are typically produced in complex microfluidic devices and, moreover, still requires stringent control to reduce droplet loss during heating. In this paper, a simple, reliable, and flexible method for fabricating PMMRAs in a 96-well plate is described in detail by using simple materials and low-cost equipment. The partitioned droplets spontaneously assemble into PMMRAs in the plates, and this distribution is maintained even after incubation. This is advantageous for in situ analysis based on an individual droplet in droplet digital loop-mediated isothermal amplification (ddLAMP) and does not require the transfer of positive droplets. Precise and reproducible quantification of classical swine fever virus (CSFV) extracts was executed in these PMMRAs to verify its availability. Our results demonstrate that the proposed approach not only provides a flexible and controllable execution scheme for droplet-based nucleic acid quantification in resource-limited laboratories but also opens new perspectives for numerous analytical and biochemical applications using droplets as versatile plastic microreactors.

Particle Self-Aligning, Focusing, and Electric Impedance Microcytometer Device for Label-Free Single Cell Morphology Discrimination and Yeast Budding Analysis.

Microfluidic electric impedance flow cytometry (IFC) devices have been applied in single cell analysis, such as cell counting, volume discrimination, cell viability, etc. A cell's shape provides specific information about cellular physiological and pathological conditions, especially in microorganisms such as yeast. In this study, the particle orientation focusing was theoretically analyzed and realized by hydrodynamics. The pulse width (passing time for the particles) of the conductance signal was used to discriminate particle shapes. Spherical and rod-shaped particles with similar volumes/lengths were differentiated by the IFC device, using the impedance pulse parameters of the events. Then, typical late-budding, early budding, and unbudded yeast cells were distinguished by the width, amplitude, and ratio of width to amplitude (R) of the impedance pulse. The pulse amplitude and the R combination gate for identifying the late-budding yeast was estimated through the statistic results. Using the gate, the late-budding rates under different conditions were calculated. Late-budding rates obtained using our method showed a high correlation (R2 = 0.83) with the manual cell counting result and represented the budding status of yeast cells under different conditions proficiently. Thus, the late-budding rate calculated using the above method can be used as a qualitative parameter to assess the reproductive performance of yeast and whether a yeast culturing environment is optimal. This IFC device and cell shape discrimination method is very simple and could be applied in the fermentation industry and other microorganisms' discrimination as a rapid analysis technique in the future.

[Development of a microenvironment test chamber for airborne microbe research].

One of the most important environmental cleanliness indicators is airborne microbe. However, the particularity of clean operating environment and controlled experimental environment often leads to the limitation of the airborne microbe research. This paper designed and implemented a microenvironment test chamber for airborne microbe research in normal test conditions. Numerical simulation by Fluent showed that airborne microbes were evenly dispersed in the upper part of test chamber, and had a bottom-up concentration growth distribution. According to the simulation results, the verification experiment was carried out by selecting 5 sampling points in different space positions in the test chamber. Experimental results showed that average particle concentrations of all sampling points reached 10 7 counts/m 3 after 5 minutes' distributing of Staphylococcus aureus, and all sampling points showed the accordant mapping of concentration distribution. The concentration of airborne microbe in the upper chamber was slightly higher than that in the middle chamber, and that was also slightly higher than that in the bottom chamber. It is consistent with the results of numerical simulation, and it proves that the system can be well used for airborne microbe research.

[Analysis and Optimization of the Temperature Retard between Sample and Air Based on Nucleic Acid Amplification System Heated by Air].

It is the main method for amplifying the specific gene to use the nucleic acid amplification system to accomplish polymerase chain reaction(PCR).The temperature retard between heat source and sample exists in the heating and cooling progresses of most nucleic acid amplification system.The retard would result in the problem that the sample would take a long time to reach the set temperature and the problem would reduce the speed of integrate reaction.Non-specific products would be created in the process of amplification when the sample cannot reach the set temperature within a certainly time and the amplified efficiency would be reduced.A miniaturization nucleic acid amplification system heated by air was designed in this study according to the principle of air-heated nucleic acid amplification system and the characteristics of the PCR instrument Smart-cycler.The heat transfer process was analyzed and the heat transfer time was calculated.The actual temperature was measured in real time,and the temperature curves were fitted.The heating time was chosen by analysis results and data fitting and the air temperature was changed,while the sample temperature was recorded.The retard between sample and air was optimized by choosing the best curve of sample temperature.The temperature retard between sample and air was reduced sharply and the required time of integrate progress is shortened to 50%.We confirmed from the amplification experiment of Listeria monocytogenes that the improved system could complete 3cycles within 4minutes,and the amplification effect was good.The amplification speed and effect could be improved effectively by optimizing the delay between sample and air.

A novel nanoparticle-based fluorescent sandwich immunoassay for specific detection of Salmonella Typhimurium.

The diseases caused by foodborne pathogens have a serious impact on human health and social stability. Conventional detection methods can involve long assay times and complex pretreatment steps, making them unsuitable for rapid, large-scale analysis of food samples. We constructed a novel nano-fluorescence sandwich immunosorbent immunoassay (nano-FSIA) to rapidly detect Salmonella Typhimurium in food, based on strong covalent binding between streptavidin and biotin. We used antibodies coupled to large particle-size fluorescent microspheres as fluorescent probes for direct quantitative analysis of S. typhimurium in milk. The optimized parameters were determined, and specificity and sensitivity were validated in phosphate-buffered saline (PBS) and milk. The results demonstrated a wide dynamic detection range for S. typhimurium (103-108 colony forming units [CFU]/mL), with the limit of detection in PBS and milk at 234 and 346 CFU/mL, respectively. The results of nano-FSIA were consistent with those of plate counts and enzyme-linked immunosorbent assays, providing an effective and promising single-bacterium counting method for the rapid detection of Salmonella.

Test Article for automation purposes.

Digital recombinase polymerase amplification (dRPA) aims to quantify the initial amount of nucleic acid by dividing nucleic acid and all reagents required for the RPA reaction evenly into numerous individual reaction units, such as chambers or droplets. dRPA turns out to be a prominent technique for quantifying the absolute quantity of target nucleic acid because of its advantages including low equipment requirements, short time consumption, as well as high sensitivity and specificity. dRPA combined with microfluidics are recognized as simple, various, and high-throughput nucleic acid quantization systems. This paper classifies the microfluidic dRPA systems over the last decade. We analyze and summarize the vital technologies of various microfluidic dRPA systems (e.g., chip preparation process, segmentation principle, microfluidic control, and statistical analysis methods), and major efforts to address limitations (e.g., prevention of evaporation and contamination, accurate initiation, and reduction of manual operation). In addition, this paper summarizes key factors and potential constraints to the success of the microfluidic dRPA to help more researchers, and possible strategies to overcome the mentioned challenges. Lastly, actual suggestions and strategies are proposed for the subsequent development of microfluidic dRPA.

Overview and Future Perspectives of Microfluidic Digital Recombinase Polymerase Amplification (dRPA).

Digital recombinase polymerase amplification (dRPA) aims to quantify the initial amount of nucleic acid by dividing nucleic acid and all reagents required for the RPA reaction evenly into numerous individual reaction units, such as chambers or droplets. dRPA turns out to be a prominent technique for quantifying the absolute quantity of target nucleic acid because of its advantages including low equipment requirements, short time consumption, as well as high sensitivity and specificity. dRPA combined with microfluidics are recognized as simple, various, and high-throughput nucleic acid quantization systems. This paper classifies the microfluidic dRPA systems over the last decade. We analyze and summarize the vital technologies of various microfluidic dRPA systems (e.g., chip preparation process, segmentation principle, microfluidic control, and statistical analysis methods), and major efforts to address limitations (e.g., prevention of evaporation and contamination, accurate initiation, and reduction of manual operation). In addition, this paper summarizes key factors and potential constraints to the success of the microfluidic dRPA to help more researchers, and possible strategies to overcome the mentioned challenges. Lastly, actual suggestions and strategies are proposed for the subsequent development of microfluidic dRPA.

A self-contained and integrated microfluidic nano-detection system for the biosensing and analysis of molecular interactions.

Traditional detection methods have shortcomings such as time-consumption and requirement of large instruments, which cannot meet the demands for on-site detection or analysis. Silicon nanowire-field-effect transistor (SiNW-FET) biosensors have the advantages of high speed, high sensitivity, strong specificity, and ease of integration. However, SiNW-FET biosensors also have some demerits: they are too sensitive, environmental factors such as light, temperature, and pH easily cause interference, and their performance uniformity needs to be calibrated in advance. In this work, we constructed a self-contained and integrated microfluidic nano-detection system containing a SiNW-FET biosensor for bio-detection and analysis. All analysis processes including liquid sample delivery, optical modulation, constant temperature control, signal amplification and data acquisition, and result display were automatically performed. In series tests including light-guided ones by analyzing various types of samples with an automatic sample injection mode, the system shows good stability and robustness. Its signal accuracy was verified using a commercial high-precision ammeter (R2 = 0.9988), too. The feasibility of the system for bio-detection was verified using simulant samples of the typical microorganism Mycobacterium tuberculosis with a limit of detection of 1.0 fg mL-1. Furthermore, the process of the binding-dissociation of antibody-protein pairs was analyzed using the system, demonstrating the potential for molecular interaction analysis. This system is highly integrated, small in size, and easy to carry, which will be developed into a portable device for on-site bio-detection and analysis of molecular interactions to enable environmental testing, medical research, food and agricultural safety, military medicine, etc.

Prediction of protein subcellular location using a combined feature of sequence.

To understand the structure and function of a protein, an important task is to know where it occurs in the cell. Thus, a computational method for properly predicting the subcellular location of proteins would be significant in interpreting the original data produced by the large-scale genome sequencing projects. The present work tries to explore an effective method for extracting features from protein primary sequence and find a novel measurement of similarity among proteins for classifying a protein to its proper subcellular location. We considered four locations in eukaryotic cells and three locations in prokaryotic cells, which have been investigated by several groups in the past. A combined feature of primary sequence defined as a 430D (dimensional) vector was utilized to represent a protein, including 20 amino acid compositions, 400 dipeptide compositions and 10 physicochemical properties. To evaluate the prediction performance of this encoding scheme, a jackknife test based on nearest neighbor algorithm was employed. The prediction accuracies for cytoplasmic, extracellular, mitochondrial, and nuclear proteins in the former dataset were 86.3%, 89.2%, 73.5% and 89.4%, respectively, and the total prediction accuracy reached 86.3%. As for the prediction accuracies of cytoplasmic, extracellular, and periplasmic proteins in the latter dataset, the prediction accuracies were 97.4%, 86.0%, and 79.7, respectively, and the total prediction accuracy of 92.5% was achieved. The results indicate that this method outperforms some existing approaches based on amino acid composition or amino acid composition and dipeptide composition.