Switch-on Fluorescence Analysis of Protease Activity with the Assistance of a Nickel Ion-Nitrilotriacetic Acid-Conjugated Magnetic Nanoparticle.

Heterogeneous protease biosensors show high sensitivity and selectivity but usually require the immobilization of peptide substrates on a solid interface. Such methods exhibit the disadvantages of complex immobilization steps and low enzymatic efficiency induced by steric hindrance. In this work, we proposed an immobilization-free strategy for protease detection with high simplicity, sensitivity and selectivity. Specifically, a single-labeled peptide with oligohistidine-tag (His-tag) was designed as the protease substrate, which can be captured by a nickel ion-nitrilotriacetic acid (Ni-NTA)-conjugated magnetic nanoparticle (MNP) through the coordination interaction between His-tag and Ni-NTA. When the peptide was digested by protease in a homogeneous solution, the signal-labeled segment was released from the substrate. The unreacted peptide substrates could be removed by Ni-NTA-MNP, and the released segments remained in solution to emit strong fluorescence. The method was used to determine protease of caspase-3 with a low detection limit (4 pg/mL). By changing the peptide sequence and signal reporters, the proposal could be used to develop novel homogeneous biosensors for the detection of other proteases.

Recent advances of nanotechnology in COVID 19: A critical review and future perspective

The global anxiety and economic crisis causes the deadly pandemic coronavirus disease of 2019 (COVID 19) affect millions of people right now. Subsequently, this life threatened viral disease is caused due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, morbidity and mortality of infected patients are due to cytokines storm syndrome associated with lung injury and multiorgan failure caused by COVID 19. Thereafter, several methodological advances have been approved by WHO and US-FDA for the detection, diagnosis and control of this wide spreadable communicable disease but still facing multi-challenges to control. Herein, we majorly emphasize the current trends and future perspectives of nano-medicinal based approaches for the delivery of anti-COVID 19 therapeutic moieties. Interestingly, Nanoparticles (NPs) loaded with drug molecules or vaccines resemble morphological features of SARS-CoV-2 in their size (60–140 nm) and shape (circular or spherical) that particularly mimics the virus facilitating strong interaction between them. Indeed, the delivery of anti-COVID 19 cargos via a nanoparticle such as Lipidic nanoparticles, Polymeric nanoparticles, Metallic nanoparticles, and Multi-functionalized nanoparticles to overcome the drawbacks of conventional approaches, specifying the site-specific targeting with reduced drug loading and toxicities, exhibit their immense potential. Additionally, nano-technological based drug delivery with their peculiar characteristics of having low immunogenicity, tunable drug release, multidrug delivery, higher selectivity and specificity, higher efficacy and tolerability switch on the novel pathway for the prevention and treatment of COVID 19. © 2022 The Author(s)

Fe Element Analysis in the Spleen Tissue by Using EDS and ICP-MS after Aminated Silica Shelled Magnetite (Fe3O4) Administered to the Tail Vein of Mice

The suppression of cytokine storm in severe coronavirus disease 2019 (COVID-19) patients can be treated with monoclonal antibody therapy against CD3 for T cell receptor inhibition. An optimized liquid phase as a CD3 antibody-magnetic nanoparticle (Ab-MNP) conjugate can inhibit the overactivation of T cells. We aim to ana-lyze the distribution of Fe in the spleen after acute administration of silica-conjugated amine magnetite (Fe3O4) nanoparticles (35 nm) delivered by intravenous injection. The Fe element distribution and concentration levels in spleen tissue were analyzed using energy dispersive spectroscopy (EDS) and inductively coupled plasma-mass spectrometry (ICP-MS). The experimental result is a difference in the concentration of Fe elements, which was 1.89×103 mg/kg in the spleen of a control mouse not administered with MNPs, whereas increases sig-nificantly to 1.93×103 mg/kg in that of a mouse administered with MNPs. Further, time kinetic analysis of bio-chemical and immunological parameters is required to confirm its suitability in bio-administration. © 2022 Journal of Magnetics.

Strategies for sensitivity enhancement of point-of-care devices

Point-of-care (POC) technology reduces the time required for diagnosis at a reduced cost to facilitate early treatment, continuous monitoring, and prevention of fatal outcomes. Biosensors are the key to the development of reliable and accurate POC devices as they are capable of detecting clinical biomarkers based on bio-recognition events. Paper-based microfluidics and lateral flow assays (LFAs) are the most commonly used techniques for the development of POC devices. Electrochemical biosensors provide high sensitivity and reproducibility in comparison to optical biosensors. Sensitivity enhancement of POC devices is imperative to lower their detection limit for improved analysis of target biomarkers at low concentrations. In this review, we have discussed the need for sensitivity enhancement in POC devices. Various sensitivity enhancement strategies such as physical, chemical, electrochemical, nanomaterial, nucleic acid, enzymatic, label-based, etc. are discussed along with numerous examples. The role of biosensors in the sensitivity enhancement of POC devices is also described herein. We have illustrated the relationship between sensitivity and the limit of detection of POC devices. Several sensitivity enhancement strategies that have been either adopted or have the potential to be realized for POC devices have been summarized in tabular form. In terms of future perspectives, the sensitivity enhancement of POC devices for the detection of important biomarkers is yet to be comprehended copiously amid the rising market for POC devices.

Comparison of electrochemical immunosensor platforms for indiscriminate SARS-CoV-2 variant detection

Since the first days of the pandemic, diagnosis of patients infected with the SARS-CoV-2 has been one of the most important parameters to control the virus. For this reason, many studies have been carried out to develop various methods for rapid and accurate diagnosis, but mutations and the occurrence of successive variants have made accurate diagnosis difficult. In this study, a screen-printed carbon electrode was used to develop magnetic nanoparticle (MNP)-based electrochemical biosensing systems that selectively detect SARS-CoV-2 virus and its variants (original, alpha, beta, and delta) in nasopharyngeal swabs. These electrodes were modified with MNPs conjugated to SARS-CoV-2 S1, S2 proteins and swab samples. Then, commercially available SARS-CoV-2-specific anti-S1 and anti-S2 antibodies and antibody cocktails purified from serum samples were applied to the surface and the performance of the platforms was compared. Analytical parameters and electrode surface characterizations were performed by electrochemical measurements after each modification step. After optimization studies of the developed biosensor platforms, the detection of limit for the antibody cocktail- based sensors were determined to be 0.53-0.75 ng/mL, while it was calculated to be 0.93-0.99 ng/mL for the anti-S1 and anti- S2-based sensors. The performance of the platforms in real nasopharyngeal swab samples (negative, original, alpha, beta, and delta variants) was evaluated and it was found that the polyclonal antibody cocktail outperformed the commercial anti-S1 and anti-S2 antibodies. As a result, polyclonal antibody cocktail, with an overall sensitivity, specificity, and accuracy of 100%, is a versatile electrochemical biosensor system for the detection of the different variants of SARS-CoV-2. We hope that the biosensor platform modified with polyclonal antibodies can be used as a potential diagnostic tool that can be applied to such epidemics in the future. Synthetic biology.

Development of a magnetic nanoparticle-based method for concentrating SARS-CoV-2 in wastewater.

Several virus concentration methods have been developed to increase the detection sensitivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater, as part of applying wastewater-based epidemiology. Polyethylene glycol (PEG) precipitation method, a method widely used for concentrating viruses in wastewater, has some limitations, such as long processing time. In this study, Pegcision, a PEG-based method using magnetic nanoparticles (MNPs), was applied to detect SARS-CoV-2 in wastewater, with several modifications to increase its sensitivity and throughput. An enveloped virus surrogate, Pseudomonas phage φ6, and a non-enveloped virus surrogate, coliphage MS2, were seeded into wastewater samples and quantified using reverse transcription-quantitative polymerase chain reaction to assess the recovery performance of the Pegcision. Neither increasing MNP concentration nor reducing the reaction time to 10 min affected the recovery, while adding polyacrylic acid as a polyanion improved the detection sensitivity. The performance of the Pegcision was further compared to that of the PEG precipitation method based on the detection of SARS-CoV-2 and surrogate viruses, including indigenous pepper mild mottle virus (PMMoV), in wastewater samples (n = 27). The Pegcision showed recovery of 14.1 ± 6.3 % and 1.4 ± 1.0 % for φ6 and MS2, respectively, while the PEG precipitation method showed recovery of 20.4 ± 20.2 % and 18.4 ± 21.9 % (n = 27 each). Additionally, comparable PMMoV concentrations were observed between the Pegcision (7.9 ± 0.3 log copies/L) and PEG precipitation methods (8.0 ± 0.2 log copies/L) (P > 0.05) (n = 27). SARS-CoV-2 RNA was successfully detected in 11 (41 %) each of 27 wastewater samples using the Pegcision and PEG precipitation methods. The Pegcision showed comparable performance with the PEG precipitation method for SARS-CoV-2 RNA concentration, suggesting its applicability as a virus concentration method.

Simple workflow to repurpose SARS-CoV-2 swab/serum samples for the isolation of cost-effective antibody/antigens for proteotyping applications and diagnosis.

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy.

One-Step, Wash-free, Nanoparticle Clustering-Based Magnetic Particle Spectroscopy Bioassay Method for Detection of SARS-CoV-2 Spike and Nucleocapsid Proteins in the Liquid Phase.

With the ongoing global pandemic of coronavirus disease 2019 (COVID-19), there is an increasing quest for more accessible, easy-to-use, rapid, inexpensive, and high-accuracy diagnostic tools. Traditional disease diagnostic methods such as qRT-PCR (quantitative reverse transcription-PCR) and ELISA (enzyme-linked immunosorbent assay) require multiple steps, trained technicians, and long turnaround time that may worsen the disease surveillance and pandemic control. In sight of this situation, a rapid, one-step, easy-to-use, and high-accuracy diagnostic platform will be valuable for future epidemic control, especially for regions with scarce medical resources. Herein, we report a magnetic particle spectroscopy (MPS) platform for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biomarkers: spike and nucleocapsid proteins. This technique monitors the dynamic magnetic responses of magnetic nanoparticles (MNPs) and uses their higher harmonics as a measure of the nanoparticles' binding states. By anchoring polyclonal antibodies (pAbs) onto MNP surfaces, these nanoparticles function as nanoprobes to specifically bind to target analytes (SARS-CoV-2 spike and nucleocapsid proteins in this work) and form nanoparticle clusters. This binding event causes detectable changes in higher harmonics and allows for quantitative and qualitative detection of target analytes in the liquid phase. We have achieved detection limits of 1.56 nM (equivalent to 125 fmole) and 12.5 nM (equivalent to 1 pmole) for detecting SARS-CoV-2 spike and nucleocapsid proteins, respectively. This MPS platform combined with the one-step, wash-free, nanoparticle clustering-based assay method is intrinsically versatile and allows for the detection of a variety of other disease biomarkers by simply changing the surface functional groups on MNPs.

Current state of diagnostic, screening and surveillance testing methods for COVID-19 from an analytical chemistry point of view.

Since December 2019, we have been in the battlefield with a new threat to the humanity known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this review, we describe the four main methods used for diagnosis, screening and/or surveillance of SARS-CoV-2: Real-time reverse transcription polymerase chain reaction (RT-PCR); chest computed tomography (CT); and different complementary alternatives developed in order to obtain rapid results, antigen and antibody detection. All of them compare the highlighting advantages and disadvantages from an analytical point of view. The gold standard method in terms of sensitivity and specificity is the RT-PCR. The different modifications propose to make it more rapid and applicable at point of care (POC) are also presented and discussed. CT images are limited to central hospitals. However, being combined with RT-PCR is the most robust and accurate way to confirm COVID-19 infection. Antibody tests, although unable to provide reliable results on the status of the infection, are suitable for carrying out maximum screening of the population in order to know the immune capacity. More recently, antigen tests, less sensitive than RT-PCR, have been authorized to determine in a quicker way whether the patient is infected at the time of analysis and without the need of specific instruments.

Homogeneous circle-to-circle amplification for real-time optomagnetic detection of SARS-CoV-2 RdRp coding sequence.

Circle-to-circle amplification (C2CA) is a specific and precise cascade nucleic acid amplification method consisting of more than one round of padlock probe ligation and rolling circle amplification (RCA). Although C2CA provides a high amplification efficiency with a negligible increase of false-positive risk, it contains several step-by-step operation processes. We herein demonstrate a homogeneous and isothermal nucleic acid quantification strategy based on C2CA and optomagnetic analysis of magnetic nanoparticle (MNP) assembly. The proposed homogeneous circle-to-circle amplification eliminates the need for additional monomerization and ligation steps after the first round of RCA, and combines two amplification rounds in a one-pot reaction. The second round of RCA produces amplicon coils that anneal to detection probes grafted onto MNPs, resulting in MNP assembly that can be detected in real-time using an optomagnetic sensor. The proposed methodology was applied for the detection of a synthetic complementary DNA of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2, also known as 2019-nCoV) RdRp (RNA-dependent RNA polymerase) coding sequence, achieving a detection limit of 0.4 fM with a dynamic detection range of 3 orders of magnitude and a total assay time of ca. 100 min. A mathematical model was set up and validated to predict the assay performance. Moreover, the proposed method was specific to distinguish SARS-CoV and SARS-CoV-2 sequences with high similarity.