3D-printed capillaric ELISA-on-a-chip with aliquoting.

Sandwich immunoassays such as the enzyme-linked immunosorbent assay (ELISA) have been miniaturized and performed in a lab-on-a-chip format, but the execution of the multiple assay steps typically requires a computer or complex peripherals. Recently, an ELISA for detecting antibodies was encoded structurally in a chip thanks to the microfluidic chain reaction (Yafia et al. Nature, 2022, 605, 464-469), but the need for precise pipetting and intolerance to commonly used surfactant concentrations limit the potential for broader adoption. Here, we introduce the ELISA-on-a-chip with aliquoting functionality that simplifies chip loading and pipetting, accommodates higher surfactant concentrations, includes barrier channels that delay the contact between solutions and prevent undesired mixing, and that executed a quantitative, high-sensitivity assay for the SARS-CoV-2 nucleocapsid protein in 4×-diluted saliva. Upon loading the chip using disposable pipettes, capillary flow draws each reagent and the sample into a separate volumetric measuring reservoir for detection antibody (70 µL), enzyme conjugate (50 µL), substrate (80 µL), and sample (210 µL), and splits washing buffer into 4 different reservoirs of 40, 40, 60, and 20 µL. The excess volume is autonomously drained via a structurally encoded capillaric aliquoting circuit, creating aliquots with an accuracy of >93%. Next, the user click-connects the assay module, comprising a nitrocellulose membrane with immobilized capture antibodies and a capillary pump, to the chip which triggers the step-by-step, timed flow of all aliquoted solutions to complete the assay in 1.5 h. A colored precipitate forming a line on a nitrocellulose strip serves as an assay readout, and upon digitization, yielded a binding curve with a limit of detection of 54 and 91 pg mL-1 for buffer and diluted saliva respectively, vastly outperforming rapid tests. The ELISA chip is 3D-printed, modular, adaptable to other targets and assays, and could be used to automate ELISA in the lab; or as a diagnostic test at the point of care with the convenience and form factor of rapid tests while preserving the protocol and performance of central laboratory ELISA.

The role of artificial cells in the fight against COVID-19: deliver vaccine, hemoperfusion removes toxic cytokines, nanobiotherapeutics lower free radicals and pCO2 and replenish blood supply.

This review concentrates on how artificial cells can contribute to helping patients with COVID-19. Artificial cells have led to mRNA vaccines with more improvements to come. Excessive cytokines in severe COVID-19 can damage organs leading to death. Artificial cell-based collodion macroporous activated charcoal adsorbent can effectively remove middle molecular weight range molecules in patients. A novel hemoperfusion device based on collodion membrane macroporous synthetic resin effectively removes cytokines and recovery in COVID-19 patients. This has been approved as an emergency treatment for COVID-19 in China, Europe, and Canada. A recent nanobiotherapeutic containing haemoglobin and up to six times the concentration of red blood cell enzymes: catalase, superoxide dismutase and carbonic anhydrase. In an animal study, this can effectively lower the damaging increase in free radicals and the removal of increased tissue pCO2. This can also help as blood substitute for the severe and critical problem of COVID-19 pandemic donor blood supply crisis.KEY MESSAGESCOVID-19 and its variants have resulted in major pandemics, severe sicknesses, and deaths around the world. COVID-19 and its variants has only started less than 3 years ago, and it is even more recently that we know more about its mechanisms, requirements, prevention, and treatment. This being the case, this is the first review on the present status and future perspectives of the use of the principle of artificial cells for COVID-19 related to vaccines, treatment, and critical donor blood supply shortage.

Fluorescent nanodiamond-based spin-enhanced lateral flow immunoassay for detection of SARS-CoV-2 nucleocapsid protein and spike protein from different variants.

SARS-CoV-2 viruses, responsible for the COVID-19 pandemic, continues to evolve into new mutations, which poses a significant threat to public health. Current testing methods have some limitations, such as long turnaround times, high costs, and professional laboratory requirements. In this report, the novel Spin-Enhanced Lateral Flow Immunoassay (SELFIA) platform and fluorescent nanodiamond (FND) reporter were utilized for the rapid detection of SARS-CoV-2 nucleocapsid and spike antigens from different variants, including wild-type (Wuhan-1), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529). The SARS-CoV-2 antibodies were conjugated with FND via nonspecific binding, enabling the detection of SARS-CoV-2 antigens via both direct and competitive SELFIA format. Direct SELFIA was performed by directly adding the SARS-CoV-2 antibodies-conjugated FND on the antigens-immobilized nitrocellulose (NC) membrane. Conversely, the SARS-CoV-2 antigen-containing sample was first incubated with the antibodies-conjugated FND, and then dropped on the antigen-immobilized NC membrane to carry out the competitive SELFIA. The results suggested that S44F anti-S IgG antibody can be efficiently used for the detection of wild-type, Alpha, Delta, and Omicron variants spike antigens. Findings were comparable in direct SELFIA, competitive SELFIA, and ELISA. A detection limit of 1.94, 0.77, 1.14, 1.91, and 1.68 ng/mL can be achieved for SARS-CoV-2 N protein, wild-type, Alpha, Delta, and Omicron S proteins, respectively, via competitive SELFIA assay. These results suggest that a direct SELFIA assay can be used for antibody/antigen pair screening in diagnosis development, while the competitive SELFIA assay can serve as an accurate quantitative diagnostic tool. The simplicity and rapidity of the SELFIA platform were demonstrated, which can be leveraged in the detection of other infectious diseases in the near future.

Proof-of-concept for speedy development of rapid and simple at-home method for potential diagnosis of early COVID-19 mutant infections using nanogold and aptamer.

The positive single-stranded nature of COVID-19 mRNA led to the low proof-reading efficacy for its genome authentication. Thus mutant covid-19 strains have been rapidly evolving. Besides Alpha, Beta, Gamma, Delta, and Omicron variants, currently, subvariants of omicron are circulating, including BA.4, BA.5, and BA.2.12.1. Therefore, the speedy development of a rapid, simple, and easier diagnosis method to deal with new mutant covid viral infection is critically important. Many diagnosis methods have been developed for COVID-19 detection such as RT-PCR and antibodies detection. However, the former is time-consuming, laborious, and expensive, and the latter relies on the production of antibodies making it not suitable for the early diagnosis of viral infection. Many lateral-flow methods are available but might not be suitable for detecting the mutants, Here we proved the concept for the speedy development of a simple, rapid, and cost-effective early at-home diagnosis method for mutant Covid-19 infection by combining a new aptamer. The idea is to use the current lateral flow Covid-19 diagnosis system available in the market or to use one existing antibody for the Lateral Flow Nitrocellulose filter. To prove the concept, the DNA aptamer specific to spike proteins (S-proteins) was conjugated to gold nanoparticles and served as a detection probe. An antibody that is specific to spike proteins overexpressed on COVID viral particles was used as a second probe immobilized to the nitrocellulose membrane. The aptamer conjugated nanoparticles were incubated with spike proteins for half an hour and tested for their ability to bind to antibodies anchored on the nitrocellulose membrane. The gold nanoparticles were visualized on the nitrocellulose membrane due to interaction between the antigen (S-protein) with both the aptamer and the antibody. Thus, the detection of viral antigen can be obtained within 2 h, with a cost of less than $5 for the diagnosis reagent. In the future, as long as the mutant of the newly emerged viral surface protein is reported, a peptide or protein corresponding to the mutation can be produced by peptide synthesis or gene cloning within several days. An RNA or DNA aptamer can be generated quickly via SELEX. A gold-labeled aptamer specific to spike proteins (S-proteins) will serve as a detection probe. Any available lateral-flow diagnosis kits with an immobilized antibody that has been available on the market, or simply an antibody that binds COVID-19 virus might be used as a second probe immobilized on the nitrocellulose. The diagnosis method can be carried out by patients at home if a clinical trial verifies the feasibility and specificity of this method.

Wearable Collector for Noninvasive Sampling of SARS-CoV-2 from Exhaled Breath for Rapid Detection.

Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.

Dissolvable sugar barriers to enhance the sensitivity of nitrocellulose membrane lateral flow assay for COVID-19 nucleic acid.

Nitrocellulose (NC) membrane can have value-added applications for lateral flow assay (LFA)-based diagnostic tools, which has great potential for the detection of pathogens, such as COVID-19, in different environments. However, poor sensitivity of the NC membrane based LFA limits its further application in many cases. Herein, we developed a facile method for LFA sensitivity enhancement, by incorporating two-sugar barrier into LFAs: one between the conjugation pad and the test line, and the other between the test line and the control line. ORF1ab nucleic acid of COVID-19 was used as the model target to demonstrate the concept on the HF120 membrane. Results show that at optimum conditions, the two sugar barrier LFAs have a detection limit of 0.5 nM, which is compared to that of 2.5 nM for the control LFA, achieving a 5-fold sensitivity increase. This low cost, easy-to-fabricate and easy-to-integrate LFA method may have potential applications in other cellulose paper-based platforms.

Carbohydrate binding module-fused antibodies improve the performance of cellulose-based lateral flow immunoassays.

Since the pandemic outbreak of Covid-19 in December 2019, several lateral flow assay (LFA) devices were developed to enable the constant monitoring of regional and global infection processes. Additionally, innumerable lateral flow test devices are frequently used for determination of different clinical parameters, food safety, and environmental factors. Since common LFAs rely on non-biodegradable nitrocellulose membranes, we focused on their replacement by cellulose-composed, biodegradable papers. We report the development of cellulose paper-based lateral flow immunoassays using a carbohydrate-binding module-fused to detection antibodies. Studies regarding the protein binding capacity and potential protein wash-off effects on cellulose paper demonstrated a 2.7-fold protein binding capacity of CBM-fused antibody fragments compared to the sole antibody fragment. Furthermore, this strategy improved the spatial retention of CBM-fused detection antibodies to the test area, which resulted in an enhanced sensitivity and improved overall LFA-performance compared to the naked detection antibody. CBM-assisted antibodies were validated by implementation into two model lateral flow test devices (pregnancy detection and the detection of SARS-CoV-2 specific antibodies). The CBM-assisted pregnancy LFA demonstrated sensitive detection of human gonadotropin (hCG) in synthetic urine and the CBM-assisted Covid-19 antibody LFA was able to detect SARS-CoV-2 specific antibodies present in serum. Our findings pave the way to the more frequent use of cellulose-based papers instead of nitrocellulose in LFA devices and thus potentially improve the sustainability in the field of POC diagnostics.