ABSTRACT
Remdesivir and apixaban have been included in the treatment guidelines of several countries for severe COVID-19 infections. To date, no analytical method has been developed for the determination of remdesivir and apixaban in plasma matrix. The main objective of this work was to develop a highly sensitive, green-adapted spectrofluorometric method for the determination of remdesivir and apixaban at the Nanoscale. Remdesivir and apixaban showed overlapping fluorescence emission spectra at 403 nm and 456 nm when excited at 246 nm and 285 nm, respectively. This overlap was resolved in two steps. The first step was synchronous fluorescence scanning of remdesivir and apixaban, and the second step was manipulation of the second-order derivative for the obtained spectra. These steps allowed complete resolution of the overlapping fluorescence spectra and selective determination of remdesivir and apixaban at 410 and 469 nm, respectively. The variables affecting the synchronous scanning of the aforementioned drugs were optimized in terms of sensitivity parameters and principles of green analytical chemistry. The described method allowed sensitive determination of remdesivir and apixaban over the concentration range of 5-200 ng/mL and 50-3000 ng/mL, respectively. The described method was validated and successfully applied for the simultaneous determination of the mentioned drugs in pure form and in spiked human plasma.
Subject(s)
COVID-19 , Humans , COVID-19 Drug Treatment , Spectrometry, Fluorescence/methodsABSTRACT
The present study demonstrates the potential of synchronous fluorescence spectroscopy and multivariate data analysis for authentication of COVID-19 vaccines from various manufacturers. Synchronous scanning fluorescence spectra were recorded for DNA-based and mRNA-based vaccines obtained through the NHS Central Liverpool Primary Care Network. Fluorescence spectra of DNA and DNA-based vaccines as well as RNA and RNA-based vaccines were identical to one another. The application of principal component analysis (PCA), PCA-Gaussian Mixture Models (PCA-GMM)) and Self-Organising Maps (SOM) methods to the fluorescence spectra of vaccines is discussed. The PCA is applied to extract the characteristic variables of fluorescence spectra by analysing the major attributes. The results indicated that the first three principal components (PCs) can account for 99.5% of the total variance in the data. The PC scores plot showed two distinct clusters corresponding to the DNA-based vaccines and mRNA-based vaccines respectively. PCA-GMM clustering complemented the PCA clusters by further classifying the mRNA-based vaccines and the GMM clusters revealed three mRNA-based vaccines that were not clustered with the other vaccines. SOM complemented both PCA and PCA-GMM and proved effective with multivariate data without the need for dimensions reduction. The findings showed that fluorescence spectroscopy combined with machine learning algorithms (PCA, PCA-GMM and SOM) is a useful technique for vaccination verification and has the benefits of simplicity, speed and reliability.
Subject(s)
COVID-19 Vaccines , COVID-19 , Humans , Spectrometry, Fluorescence/methods , Reproducibility of Results , COVID-19/prevention & control , DNA , RNA, MessengerABSTRACT
As new infectious mutations of SARS-CoV-2 emerged throughout the world, innovative therapies to counter the virus-altered drug sensitivities were urgently needed. Several antiviral options have been in clinical trials or in compassionate use for the treatment of SARS-CoV-2 infections in an attempt to minimize both clinical severity and viral shedding. Recent research indicated that simeprevir acts synergistically with remdesivir, allowing for a multiple-fold decrease in its effective dose when used at physiologically acceptable concentrations. The goal of this work is to develop a sensitive synchronous spectrofluorimetric approach to simultaneously quantify the two drugs in biological fluids. Using this method, remdesivir and simeprevir could be measured spectrofluorimetrically at 283 and 341 nm, respectively, without interference from each other using Δλ of 90 nm. The effect of various experimental parameters on the fluorescence intensity of the two drugs was extensively explored and optimized. For each of remdesivir and simeprevir, the method exhibited a linearity range of 0.10-1.10 µg/mL, with lower detection limits of 0.01 and 0.02 µg/mL and quantification limits of 0.03 and 0.05 µg/mL, respectively. The high sensitivity of the developed method permitted the simultaneous determination of both drugs in spiked plasma samples with % recoveries ranging from 95.0 to 103.25 with acceptable standard deviation values of 1.92 and 3.04 for remdesivir and simeprevir, respectively. The validation of the approach was approved by the International Council of Harmonization (ICH) guidelines.
Subject(s)
COVID-19 , Simeprevir , Humans , SARS-CoV-2 , COVID-19 Drug Treatment , Antiviral Agents , Spectrometry, Fluorescence/methodsABSTRACT
We report the development of a label-, antibody-, enzyme-, and amplification-free ratiometric fluorescent biosensor for low-cost and rapid (less than 12 min) diagnosis of COVID-19 from isolated RNA samples. The biosensor is designed on the basis of cytosine-modified antisense oligonucleotides specific for either N gene or RdRP gene that can form silver nanoclusters (AgNCs) with both green and red emission on an oligonucleotide via a one-step synthesis process. The presence of the target RNA sequence of SARS-CoV-2 causes a dual-emission ratiometric signal transduction, resulting in a limit of detection of 0.30 to 10.0 nM and appropriate linear ranges with no need for any further amplification, fluorophore, or design with a special DNA fragment. With this strategy, five different ratiometric fluorescent probes are designed, and how the T/C ratio, the length of the stem region, and the number of cytosines in the loop structure and at the 3' end of the cluster-stabilizing template can affect the biosensor sensitivity is investigated. Furthermore, the effect of graphene oxide (GO) on the ratiometric behavior of nanoclusters is demonstrated and the concentration-/time-dependent new competitive mechanism between aggregation-caused quenching (ACQ) and aggregation-induced emission enhancement (AIE) for the developed ssDNA-AgNCs/GO nanohybrids is proposed. Finally, the performance of the designed ratiometric biosensor has been validated using the RNA extract obtained from more than 150 clinical samples, and the results have been confirmed by the FDA-approved reverse transcription-polymerase chain reaction (RT-PCR) diagnostic method. The diagnostic sensitivity and specificity of the best probe is more than >90%, with an area under the receiver operating characteristic (ROC) curve of 0.978.
Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , Humans , Fluorescent Dyes/chemistry , Silver/chemistry , Metal Nanoparticles/chemistry , COVID-19/diagnosis , SARS-CoV-2/genetics , DNA , RNA , Biosensing Techniques/methods , Spectrometry, Fluorescence/methodsABSTRACT
COVID-19 is a fast-spreading pandemic that is caused by SARS-CoV-2 viral pathogen. Combination therapy of the antiviral favipiravir and the anticoagulant apixaban is one of the efficient treatment regimens. Therefore, development of novel and sensitive methods for simultaneous analysis of such combination is highly advantageous. Herein, two eco-friendly, simple, rapid, and cost-effective spectrofluorometric methods were evolved for the estimation of favipiravir and apixaban in pharmaceutical and biological matrices. Method I was based on analysis of favipiravir and apixaban by the first-order derivative of the conventional fluorescence spectra obtained after excitation at 300 nm, where favipiravir and apixaban were detected at 468.8 and 432.0 nm, respectively. Method II relied on dual scan synchronous spectrofluorometry, in which favipiravir was determined at 364 nm using Δλ = 60 nm while apixaban was analyzed at 274 nm using Δλ = 200 nm. Method optimization was performed for selecting the optimum conditions at which maximum sensitivity and selectivity were obtained. This report is the first one that describes simultaneous analysis of favipiravir and apixaban by synchronous spectrofluorometry. The developed methods were successfully applied to evaluate favipiravir and apixaban in spiked human plasma and in pharmaceutical dosages with high %recoveries and low RSD.
Subject(s)
COVID-19 , Humans , Spectrometry, Fluorescence/methods , SARS-CoV-2 , Amides , Antiviral Agents/therapeutic use , Pharmaceutical PreparationsABSTRACT
Few reports are found working on the features and functions of the human telomere G-triplex (ht-G3) though the telomere G-quadruplex has been intensely studied and widely implemented to develop various biosensors. We herein report that ht-G3 lights up Thioflavin T (ThT) and establish a sensitive biosensing platform for RNA detection by introducing a target recycling strategy. An optimal condition was selected out for ht-G3 to promote ThT to generate a strong fluorescence. Accordingly, an ht-G3-based molecular beacon was successfully designed against the corresponding RNA sequence of the SARS-CoV-2 N-gene. The sensitivity for the non-amplified RNA target achieves 0.01 nM, improved 100 times over the conventional ThT-based method. We believe this ht-G3/ThT-based label-free strategy could be widely applied for RNA detection.
Subject(s)
Biosensing Techniques , COVID-19 , G-Quadruplexes , Benzothiazoles , Biosensing Techniques/methods , DNA/genetics , Fluorescent Dyes , Humans , Limit of Detection , RNA , SARS-CoV-2 , Spectrometry, Fluorescence/methods , TelomereABSTRACT
Following the sudden widespread of the novel coronavirus (COVID-19) which first appeared in Wuhan city. Remdesivir (REM) was the first medicine licensed by the US Food and Drug Administration (FDA) for COVID-19 infected hospitalized patients. Hence, there was an urgent demand for the optimization of efficient selective and sensitive methods to be developed for the determination of REM in pharmaceuticals as well as biological samples. A sensitive and simple green spectrofluorimetric method has been developed to determine REM in pharmaceutical formulation, in addition to, spiked human plasma. The technique involves measuring the native fluorescence of REM in distilled water at 410 nm followed by excitation at 241 nm, giving a linear relationship over the range 50.00-500.00 ng/mL, and then improving the sensitivity of REM through micellar formation using 2.00% w/v sodium dodecyl sulfate (SDS). A linear relationship has been obtained over the range 10.00-350.00 ng/mL having detection and quantitation limits of 2.34 and 7.10 ng/mL, respectively. Different analytical parameters have been carefully studied. A validation study has been conducted successfully in accordance with the FDA and the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The developed methods' greenness was assessed utilizing a greenness profile and analytical eco-scale standards. Both methods were discovered to be environmentally friendly and could be successfully used for the determination of the studied drugs in pharmaceutical formulation and human plasma with good accuracy and high precision. As a result, the developed spectrofluorimetric methods could be ideally suited for determination of REM in quality control and medicinal laboratories.
Subject(s)
COVID-19 Drug Treatment , Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Humans , Micelles , Spectrometry, Fluorescence/methodsABSTRACT
Internalization of membrane proteins plays a key role in many physiological functions; however, highly sensitive and versatile technologies are lacking to study such processes in real-time living systems. Here we describe an assay based on bioluminescence able to quantify membrane receptor trafficking for a wide variety of internalization mechanisms such as GPCR internalization/recycling, antibody-mediated internalization, and SARS-CoV2 viral infection. This study represents an alternative drug discovery tool to accelerate the drug development for a wide range of physiological processes, such as cancer, neurological, cardiopulmonary, metabolic, and infectious diseases including COVID-19.
Subject(s)
Drug Discovery/methods , Membrane Proteins , Protein Transport/physiology , Spectrometry, Fluorescence/methods , COVID-19 , Drug Development/methods , HEK293 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Microscopy, Fluorescence , Nanotechnology , Receptors, G-Protein-Coupled , SARS-CoV-2/chemistry , SARS-CoV-2/metabolism , Virus InternalizationABSTRACT
The interactions of four sulfonylated Phe(3-Am)-derived inhibitors (MI-432, MI-463, MI-482 and MI-1900) of type II transmembrane serine proteases (TTSP) such as transmembrane protease serine 2 (TMPRSS2) were examined with serum albumin and cytochrome P450 (CYP) isoenzymes. Complex formation with albumin was investigated using fluorescence spectroscopy. Furthermore, microsomal hepatic CYP1A2, 2C9, 2C19 and 3A4 activities in presence of these inhibitors were determined using fluorometric assays. The inhibitory effects of these compounds on human recombinant CYP3A4 enzyme were also examined. In addition, microsomal stability assays (60-min long) were performed using an UPLC-MS/MS method to determine depletion percentage values of each compound. The inhibitors showed no or only weak interactions with albumin, and did not inhibit CYP1A2, 2C9 and 2C19. However, the compounds tested proved to be potent inhibitors of CYP3A4 in both assays performed. Within one hour, 20%, 12%, 14% and 25% of inhibitors MI-432, MI-463, MI-482 and MI-1900, respectively, were degraded. As essential host cell factor for the replication of the pandemic SARS-CoV-2, the TTSP TMPRSS2 emerged as an important target in drug design. Our study provides further preclinical data on the characterization of this type of inhibitors for numerous trypsin-like serine proteases.
Subject(s)
Antiviral Agents/metabolism , Cytochrome P-450 Enzyme System/metabolism , Protease Inhibitors/metabolism , Serine Endopeptidases/metabolism , Serum Albumin, Human/metabolism , Antiviral Agents/analysis , Antiviral Agents/pharmacology , Dose-Response Relationship, Drug , Humans , Isoenzymes/metabolism , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Protease Inhibitors/analysis , Protease Inhibitors/pharmacology , Protein Binding/physiology , Serine Endopeptidases/analysis , Spectrometry, Fluorescence/methods , Tandem Mass Spectrometry/methodsABSTRACT
This paper describes a detailed study of spectral and time-resolved photoprocesses in human platelets and their complexes with platinum (Pt) nanoparticles (NPs). Fluorescence, quantum yield, and platelet amino acid lifetime changes in the presence and without femtosecond ablated platinum NPs have been studied. Fluorescence spectroscopy analysis of main fluorescent amino acids and their residues (tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe)) belonging to the platelet membrane have been performed. The possibility of energy transfer between Pt NPs and the platelet membrane has been revealed. Förster Resonance Energy Transfer (FRET) model was used to perform the quantitative evaluation of energy transfer parameters. The prospects of Pt NPs usage deals with quenching-based sensing for pathology's based on platelet conformations as cardiovascular diseases have been demonstrated.
Subject(s)
Blood Platelets/chemistry , Fluorescence Resonance Energy Transfer/methods , Metal Nanoparticles/chemistry , Platinum/chemistry , Adult , Energy Transfer , Healthy Volunteers , Humans , Spectrometry, Fluorescence/methodsABSTRACT
Heparan sulfate (HS), a sulfated glycosaminoglycan (GAG), was reported to be a necessary host attachment factor that promotes SARS-CoV-2 infection. In this study, we developed GAG microarrays based on fluorescence detection for high-sensitivity screening of the GAG-binding specificity of proteins and applied it for the analysis of SARS-CoV-2 spike (S) protein. Among the 20 distinct GAGs, the S protein bound not only to heparin (HEP)/HS but also to chondroitin sulfate E (CSE) in a concentration-dependent manner. We then analyzed the specificity of each subunit of the S protein. While the S1 subunit showed exclusive binding to HEP, the S2 subunit also bound to CSE and HEP/HS. CSE might act as an alternative attachment factor for HS in SARS-CoV-2 infection.
Subject(s)
Chondroitin Sulfates/metabolism , Glycosaminoglycans/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Humans , Microarray Analysis , Protein Binding , Spectrometry, Fluorescence/methodsABSTRACT
Exponential molecular amplification such as the polymerase chain reaction is a powerful tool that allows ultrasensitive biodetection. Here, we report a new exponential amplification strategy based on photoredox autocatalysis, where eosin Y, a photocatalyst, amplifies itself by activating a nonfluorescent eosin Y derivative (EYH3-) under green light. The deactivated photocatalyst is stable and rapidly activated under low-intensity light, making the eosin Y amplification suitable for resource-limited settings. Through steady-state kinetic studies and reaction modeling, we found that EYH3- is either oxidized to eosin Y via one-electron oxidation by triplet eosin Y and subsequent 1e-/H+ transfer, or activated by singlet oxygen with the risk of degradation. By reducing the rate of the EYH3- degradation, we successfully improved EYH3--to-eosin Y recovery, achieving efficient autocatalytic eosin Y amplification. Additionally, to demonstrate its flexibility in output signals, we coupled the eosin Y amplification with photoinduced chromogenic polymerization, enabling sensitive visual detection of analytes. Finally, we applied the exponential amplification methods in developing bioassays for detection of biomarkers including SARS-CoV-2 nucleocapsid protein, an antigen used in the diagnosis of COVID-19.
Subject(s)
Coronavirus Nucleocapsid Proteins/analysis , Eosine Yellowish-(YS)/analogs & derivatives , Spectrometry, Fluorescence/methods , 3,3'-Diaminobenzidine/chemistry , Biomarkers/chemistry , Catalysis/radiation effects , Eosine Yellowish-(YS)/chemical synthesis , Eosine Yellowish-(YS)/radiation effects , Fluorescence , Light , Limit of Detection , Oxidation-Reduction/radiation effects , Phosphoproteins/analysis , Polyethylene Glycols/chemistry , Polymerization , Proof of Concept Study , SARS-CoV-2/chemistryABSTRACT
The present work describes development of rapid, robust, sensitive and green spectrofluorimetric method for determination of favipiravir (FAV). Different factors affecting fluorescence were carefully studied and Box Behnken Design was applied to optimize experimental parameters. The proposed method is based on measuring native fluorescence of FAV in 0.2 M borate buffer (pH 8.0) at 432 nm after excitation at 361 nm. There was a linear relationship between FAV concentration and relative fluorescence intensity over the range 40-280 ng/mL with limit of detection of 9.44 ng/mL and quantitation limit of 28.60 ng/mL. The method was successfully implemented for determination of FAV in its pharmaceutical formulation with mean % recovery of 99.26 ± 0.87. Moreover, the high sensitivity of the method allowed determination of FAV in spiked human plasma over a range of 48-192 ng/mL. The proposed spectrofluorimetric method was proved to be eco-friendly according to analytical eco-scale.
Subject(s)
Amides/blood , Antiviral Agents/blood , COVID-19 Drug Treatment , COVID-19/blood , Pyrazines/blood , Spectrometry, Fluorescence/methods , Amides/analysis , Amides/therapeutic use , Antiviral Agents/analysis , Antiviral Agents/therapeutic use , Blood Chemical Analysis/methods , Blood Chemical Analysis/statistics & numerical data , Humans , Limit of Detection , Pyrazines/analysis , Pyrazines/therapeutic use , SARS-CoV-2 , Sensitivity and Specificity , Spectrometry, Fluorescence/statistics & numerical dataABSTRACT
The proliferation and transmission of viruses has become a threat to worldwide biosecurity, as exemplified by the current COVID-19 pandemic. Early diagnosis of viral infection and disease control have always been critical. Virus detection can be achieved based on various plasmonic phenomena, including propagating surface plasmon resonance (SPR), localized SPR, surface-enhanced Raman scattering, surface-enhanced fluorescence and surface-enhanced infrared absorption spectroscopy. The present review covers all available information on plasmonic-based virus detection, and collected data on these sensors based on several parameters. These data will assist the audience in advancing research and development of a new generation of versatile virus biosensors.