The current outbreak of pandemic coronavirus disease 2019 (COVID-19) aggravates serious need for effective therapeutics. Over recent years, drug repurposing has been accomplished as an important opportunity in drug development as it shortens the time consumed for development, besides sparing the cost and the efforts exerted in the research and development process.in vitro inhibition of SARS-CoV-2 at a small micromolar concentration. Additionally, NTZ suppresses the production of cytokines emphasizing its potential to manage COVID-19-induced cytokine storm. Furthermore, the reported efficacy of NTZ to bronchodilate the extremely contracted airways can be beneficial in alleviating COVID-19-associated symptoms.The FDA-approved antiparasitic drug, nitazoxanide (NTZ), has been found to have antiviral activity against different viral infections such as coronaviruses, influenza, hepatitis C virus (HCV), hepatitis B virus (HBV), and other viruses signifying its potential as a broad spectrum antiviral drug. Moreover, it has been recently reported that NTZ exhibited All these findings, along with the high safety record of the drug, have gained our interest to urge conductance of clinical trials to assess the potential benefits of using it in COVID-19 patients. Thus, in this summarized article, we review the antiviral activities of NTZ and highlight its promising therapeutic actions that make the drug worth clinical trials. Cryptosporidium spp. and Giardia lamblia in 2002  and one influenza B virus (IBV) [Influenza B/Parma/3/04]. The half maximal inhibitory concentration of TIZ against influenza viruses ranged from 0.2 to 1.5 μg/ml [NTZ readily undergoes hydrolysis and converts to TIZ in aqueous buffers as a cell culture medium , 29. Sev.5 μg/ml , and it .5 μg/ml . Moreove.5 μg/ml .Tilmanis et al. have reported the potent antiviral effect of TIZ against 210 seasonal influenza viruses as well as viruses that exhibit resistance to neuraminidase inhibitors including oseltamivir, zanamivir, peramivir, and laninamivir .p = 0.0006). Interestingly, no observable antiviral resistance against NTZ was reported among treated influenza viruses and no adverse events were reported on humoral immune response in the drug-receiving patients [In a human clinical trial involving 257 patients, NTZ could effectively reduce the duration of symptoms of influenza in patients that received oral doses of 600 mg twice daily comparing to the placebo. Additionally, there was a significant decrease in the viral titers in this group of patients when compared to patients that received placebo  [Results of a randomized, 2-phase, double-blind clinical study which was conducted enrolling 200 subjects (including 50 adults and 150 children from 2 months to 11 years old) with viral gastroenteritis induced by the infection with norovirus or rotavirus signified the ability of NTZ for the significant reduction in the duration of disease symptoms when compared with the placebo ( 0.0295) . Morris  0.0295) .Dang et al. have reported a synergistic inhibitory effect of human norovirus replication and complete depletion of the replicons from the host cells after administration of combination therapy of ribavirin and TIZ. Briefly, their results revealed that TIZ inhibits viral replication by stimulation of cellular antiviral response particularly through expression of interferon regulatory factor 1. Treatment with NTZ alone or in combination with ribavirin represents a promising strategy for combating norovirus-induced gastroenteritis principally in immunosuppressed patients .in vitro inhibition of the replication of several astrovirus strains as well as in vivo reduction in the viral shed and diarrhea in symptomatic astrovirus-infected turkey poultry model [50 of 1.47 μM.Human astrovirus is known to cause 2 to 9% of acute non-bacterial diarrhea pediatric cases globally. Human astrovirus infections can be fatal particularly in immunocompromised patients as the infection may be associated with necrotizing enterocolitis and severe diarrhea, in addition to other fatal diseases such as respiratory diseases , encephary model . Their rHuman adenoviruses are non-enveloped, double-stranded DNA viruses with icosahedral capsids. Infection with human adenovirus usually leads to severe life-threatening disease particularly in immunocompromised subjects. At the moment, there is a lack of available specific antiviral drugs against adenovirus infections . GarrigoParamyxoviruses are a group of airborne viruses which are responsible for some respiratory diseases. They include agents of measles, mumps, respiratory syncytial virus, Newcastle disease in poultry, and parainfluenza in human. These viruses are characterized by single-stranded RNA genome and polymerase. They have an envelope of a diameter between 150 and 300 nm. The viruses had been reported in some species of snakes which is transmitted to human . They arPiacentini et al. identified in their study the anti-paramyxovirus effect of NTZ; they found that NTZ halts the F-trafficking to the plasma by acting on Sendai virus and RSV F-protein aggregate of the viral cell. They further found NTZ acting as an ERp57 non-competitive blocker; the NTZ binding site is said to be located at the ERp57-b/b′ non-catalytic domain border .Ebola virus is an enveloped negative single-stranded RNA virus. Ebola virus is transmitted to human through wild animals, and it can further spread between humans via transmission from subject to subject. Ebola virus infection is identified as Ebola hemorrhagic fever; it exhibited high mortality rates (25 to 90% in the previous outbreaks) . Jasenos50 for genotype 1a was 0.09 μg/ml, while for genotype 1b, it was 0.06 μg/ml. Synergism was also reported upon combining TIZ with interferon α or with 2′C methylcytidine [HCV is an enveloped positive-sense single-stranded RNA virus belonging to the family Flaviviridae. The virus can be transmitted essentially through blood to cause either acute or chronic hepatitis. HCV is encountered as the essential cause of hepatic cancer . TIZ actcytidine . It is ncytidine .Clinical trials were conducted to assess the efficacy of NTZ against HCV with promising outcomes as patients accomplished a sustained virological response without traceable viral RNA in serum after NTZ therapy , 48. Evein vitro study showed that NTZ acts by leading HBsAg loss by restraining serum HBV DNA. NTZ shows an original mechanism of antivirals with the inhibition of oxidative phosphorylation in the mitochondria [HBV is a source of significant progressive liver disease; an estimated number of two billion people are living with HBV in the world, out of which approximately 360 million people are infected chronically . Korba echondria . Furtherchondria .Chikungunya virus is a positive-sense single-stranded RNA virus. Chikungunya virus causes chikungunya disease that is identified by fever, conjunctivitis, arthritis, and severe arthralgia. The infection is transmitted by the female mosquitoes infected with the virus. In 2005, the outbreak of the disease occurred in France, and then, the infection spread to many other countries with hundreds of deaths and greater than one million infected cases . Chikung50 of 0.35 μg/ml [Rubella virus is characterized as a single-stranded positive RNA genome. It is small and enveloped . The dis35 μg/ml .50 of 2 μM [Vaccinia virus is utilized as a vector for the delivery of the other antigens . Neverth of 2 μM .50 was about 0.5 μg/ml). Additionally, NTZ exhibited synergistic antiviral effects when combined with some HIV drugs including inhibitors of integrase , nucleoside, and non-nucleoside reverse transcription namely azidothymidine and efavirenz, respectively [HIV infection is devastating to humans since 30 years as a minimum. Since then, HIV infected 60 million subjects and caused more than 25 million deaths. Highly active antiretroviral therapy is available and effective treatment strategy for acquired immune deficiency patients; this strategy typically includes a combination of three drugs from two or more classes. For instance, two drugs belonging to the class of nucleoside reverse transcriptase inhibitors are combined with a third drug from these classes: integrase inhibitors, non-nucleoside reverse transcriptase inhibitors, or protease inhibitors. As a result of the high cost of the currently available drugs and the potential serious side effects, development of new antiviral drugs is needed . Tan et ectively .50 of 3.2 μM. These findings signified a promising antiviral effect of NTZ that is needed to treat congenital infections and the development of strains that can resist the current anti-DNA polymerase compounds [More than 60% of human population is susceptible to human cytomegalovirus infections globally. The highly risk group of population includes acquired immune deficiency patients and immunocompromised subjects since the significances of cytomegalovirus infections may cause severe and life-threatening complications . Mercoreompounds .50 of 0.12 μg/mL; in vivo activity of NTZ was also studied on mice infected with a fatal dose of Japanese encephalitis virus, and the results revealed that the infected mice could survive after they received NTZ orally for a period of 25 days. On the other hand, all untreated mice died on the ninth day of the experiment. The survival of the mice was proportional to the administered doses as the observed survival was 30%, 70%, and 90% for NTZ doses of 50, 75, and 100 mg/kg/day, respectively [TIZ could effectively inhibit the replication of both dengue-2 and yellow fever in Vero cell lines with half maximal inhibitory concentrations of 0.1 and 0.06 μg/ml, respectively . AnotherBesides the antiviral activity of NTZ, the ability of NTZ to inhibit pro-inflammatory cytokine production including TNFα, interleukin (IL)-2, IL-4, I-5, IL-6, IL-8, and IL-10 in PBMC  was also reported . Other s+2-activated Cl− channel (TMEM16A) antagonist that causes blockade in the contraction and depolarization of the airway smooth muscles. They assessed NTZ efficacy in harsh conditions utilizing extremely contracted airways or airways that were subjected to a cytokine cocktail, and the results revealed that the drug could surprisingly bronchodilate airways. Their findings suggest the potential benefit of using NTZ in COVID-19 patients as it may help to alleviate the disease-associated symptoms [In a recent study, Miner and his colleagues reported that NTZ is a potent Casymptoms .50 signifying that the standard dosing regimen of NTZ will be adequate to maintain effective antiviral concentrations throughout the treatment. A published study reported that the administration of a high single dose (4 g) of NTZ achieved a maximum plasma concentration of approximately 200 μM in patients and even this high dose was well tolerated without toxicity [50 against SARS-CoV-2 is only 2.12 μM, and that can be easily achieved with the typical dosing of NTZ.NTZ exhibits extraordinary broad spectrum antimicrobial activity . In the toxicity . These d50 values of 2.12 μM, 1.13 μM, and 0.77 μM, respectively. It is reported in a Chinese study that oseltamivir, which is a drug that is commonly used for influenza A and B, had no positive effects as a treatment for COVID-19 [in vitro inhibitory effect against SARS-CoV-2 within 48 h with an IC50 of approximately 2 μM [The COVID-19 outbreak is considered as a threat for public health and a global emergency to search for novel effective treatment options to hinder the rapid spreading of the lethal COVID-19 which is urgently necessitated. Drug repurposing campaigns can reduce the cost and time required for developing new molecules. It is recently reported that some screened current drugs such as NTZ, chloroquine, and remdesivir were proved to inhibit the fatal SARS-CoV-2 using low concentrations in cultured Vero E6 cell lines with ICCOVID-19 . HoweverCOVID-19 . Additioely 2 μM . There aely 2 μM . Moreoveely 2 μM . Furtherely 2 μM . Althougely 2 μM , this auely 2 μM . Newly din vitro inhibitory action of NTZ against SARS-CoV-2 and other coronaviruses with easily attainable concentration, (ii) the inhibitory effect against pro-inflammatory cytokines including the suppression of IL-6 production, (iii) bronchodilatory effect, and (iv) a satisfactory safety record verified by the clinical trials and in extensive post-marketing experience.In conclusion, the available reported data suggest the potential therapeutic effects of NTZ in the treatment of SAR-CoV-2 with particular emphasis on (i)


We are disoriented. The most current reason for this is the COVID-19 pandemic, but our disorientation actually started long before. Let’s take the expression We as simply meaning “humankind”, and COVID-19 for the interaction between viruses and mammals, including humans. Right now We are being strongly influenced by global phenomena: The disorientation We've already experienced due to global warming, virtual transformation and the expansion of the technosphere has been increased by the addition of another form, i.e. the immediate disorientation caused by a virus.We are stuck right in the middle, across borders—and responsible. When it comes to climate change We have always been (and still are) caught up in a discussion about whether We are already a part of it, but the coronavirus leaves no doubt about this: not in the way it acts or how enters our bodies, nor in the way it spreads globally. The virus ignores and transcends organic, continental and political boundaries. At the same time it closes borders; images of entire fleets of aircraft on the ground and travel bans are examples of this. These are indeed paradoxical phenomena, which exacerbate our disorientation and force us to immediately deal with an unknown. From a systemic perspective, it could be said that the virus unintentionally uses us as a system chain, with the result being that We have broken many other system chains that were considered untouchable up to now.The dissolving of boundaries and the questioning of existing categories and systematics has preoccupied us for some time now. With the beginning of the new century and the concept of the Anthropocene, these issues have gained enormous momentum, both in terms of real life and scientific theory. And now, within a matter of weeks, the virus has become a marker, accelerator and spokesperson for the Anthropocene discourse.We—are believed to have become a geological factor. The concept of the Anthropocene (Crutzen What does the Anthropocene imply? It expresses the idea, in short, that the changes in the Earth system caused by humans are comparable to geological forces. Humans themselves—the global  Crutzen  has been Crutzen . It is uWe can now directly and physically experience the Anthropocene changes of a natural system that was long considered stable. The corona virus has forced us to quickly broaden our perspective. We are becoming conscious of the fact that the spread of the human species and its way of living is not only of a geological magnitude. Due to the spread of human-influenced habitats, the interaction between humans and animals is also being intensified, and thus, according to virologists, viruses can more easily change from animal hosts to humans. Just under half of the viral species found in mammals have so far been detected in humans, and most of them are a result of zoonosis.With regards to climate change, The causes that have led to these intensified human-animal interactions are manifold, and they go hand in hand with our globalised, neoliberal way of life and economy. Population growth and migration, as well as the restriction of habitats for wild animals through an expansion of human activities are cited by scientist as reasons, as are the intensification of agriculture, changes in our food system, a mobility pattern that has lead to more intercontinental travel, a public health system that is desolate in many areas, and global warming.We proceed?So how do One consequence for the disciplines that design space must be on a higher level and fundamentally question the relationship between the co-existence of humans and other species and space Renn . While iWe should regard the present situation as an indispensable challenge to radically broaden our frame of thought and to orient and apply our thinking and acting to the creation of connections. Where do We stand? Isn’t it absurd that the amount of chicken bones produced worldwide—which is breathtakingly high—was actually discussed as a marker for the scientific classification of the Anthropocene (Bennett et al. We co-exist with non-human species in the future? Are dairy cows merely a type of technology for us (Gan et al. Technology and animals should be our family today, demands the feminist, biologist and utopian thinker Haraway  in a verWe face other urgent issues as well: How do We protect ourselves, how do We slow down the spread of the virus, and how do We show solidarity in the fight against this virus? If We want to achieve co-existence, We must take a different long-term view of our vulnerability. Is there a We at all?At the current stage of being directly affected by the virus, COVID-19 also ultimately reminds us of the manifold unsolved challenges of the porosity and closure of borders between different species, humans, animals and viruses, and of the organisation of space associated with them. It makes us aware of the daily mutual pervasiveness and linking of systems and system chains, intended or not. In questions of nutrition, everyday each of us is, in dependence on the global food system, a contributor and decision maker regarding our direct links to non-human species. This virus makes it clear to us that the times of uninhibited consumption are finally over, and that more space, both mentally and physically, must be given to questions of co-existence.


Magnetically actuated lab-on-a-chip (LOC) technologies have enabled rapid, highly efficient separation of specific biomarkers and cells from complex biological samples. Nonlinear magnetophoresis (NLM) is a technique that uses a microfabricated magnet array (MMA) and a time varying external magnetic field to precisely control the transport of superparamagnetic (SPM) beads on the surface of a chip based on their size and magnetization. We analyze the transport and separation behavior of SPM monomers and dimers on four MMA geometries, i.e., circular, triangular, square and rectangular shaped micromagnets, across a range of external magnetic field rotation frequencies. The measured critical frequency of the SPM beads on an MMA, i.e., the velocity for which the hydrodynamic drag on a bead exceeds the magnetic force, is closely related to the local magnetic flux density landscape on a micromagnet in the presence of an external magnetic field. A set of design criteria has been established for the optimization of MMAs for NLM separation, with particular focus on the shape of the micromagnets forming the array. The square MMA was used to detect a model protein biomarker and gene fragment based on a magnetic bead assembly (MBA) assay. This assay uses ligand functionalized SPM beads to capture and directly detect an analyte through the formation of SPM bead aggregates. These beads aggregates were detected through NLM separation and microscopic analysis resulting in a highly sensitive assay that did not use carrier fluid. Magnetophoretic LOC research initially focused on miniaturizing high gradient magnetic field separation due to the central role SPM beads play in in vitro diagnostics4, and has rapidly expanded to include a number of functions, including simultaneous detection of multiple analytes7. Significantly, on-chip detection technologies have been developed to count SPM beads using giant magnetoresistance8, tunneling magnetoresistance9, planar Hall magnetoresistance10, and optical sensing11. The integration of high gradient separation with these sensors promises to deliver devices that are capable of rapid and sensitive point of care testing (POCT)14.Superparamagnetic (SPM) microparticles have been used to efficiently separate nucleic acids, proteins, viruses, and cells from complex samples for lab-on-a-chip (LOC) sensing, e.g., SPM beads functionalized with antibodies can be used to isolate as few as 100 copies of a virus from a milliliter of serum sample in 2 min16. A key feature of SPM microparticles is that particle–particle interactions are minimal in the absence of a significant applied magnetic field and thus rapid rates of mass transfer and reaction take place in dense microparticle suspensions17. Nonmagnetic particle aggregation induced by cross-reaction does not take place rapidly due to the repulsive hydrodynamic force inherent in these suspensions. In the magnetic bead assembly (MBA) assay, a high gradient magnetic field is used to link two or more SPM beads through a specific analyte using the appropriate chemistries, as shown for model protein and nucleic acid systems in Fig. 19, light scattering21, flow cytometry2 and reflectometry16. The isolation of the analyte carrying SPM beads could improve the performance of magnetic LOC assays by increasing the total number of beads that can be employed for analysis, facilitating the detection of low concentrations of analyte, and enabling the recovery of the target for further processing.Single-step detection of an analyte can be achieved by monitoring the assembly of SPM beads25. Figure xz = 0° to − 270°). The magnetic force focuses the SPM beads to the point of maximum flux density and thus they travel across the array at a speed defined by the frequency of rotation of the external field. A simplified one-dimensional transport model has been used to describe the motion of SPM beads based on the magnetic force and hydrodynamic drag forces26. Significant improvements have been made to modelling the transport behavior of SPM beads on micromagnets by taking into account their hydrodynamic interaction with the substrate but are beyond the scope of this article29. When the rotational frequency of the external field (ω) is slow the beads are transported across the micromagnets at a constant velocity equal (v) in the ‘phase-locked’ regime d is the center-to-center distance between adjacent micromagnets. Above a critical frequency, ωc, the beads begin to slip with respect to the translating magnetic flux density landscape due to the magnitude of the hydrodynamic force. The critical frequency has a dependence on the characteristic properties of the SPM beads and MMA.χ is the beads’ magnetic susceptibility, μo is a constant denoting the magnetic permeability of vacuum, σo is an experimentally determined parameter that describes the field distribution generated by the micromagnets, Hext is the magnetic field, η is the viscosity of the surrounding medium and β = r/d (where r is the radius of the bead). The slipping is observed as a rocking motion between adjacent micromagnets superimposed on the time-averaged linear velocity, which becomes zero at high frequencies. In the ‘phase slipping’ regime the velocity of the beads is approximately.Precise control of the transport of SPM beads can be achieved through the combination of a rotating magnetic field with the microfabricated magnet arrays (MMA)31. NLM allows the transport of high densities of SPM beads, i.e., ca. 105 micromagnets per cm2, with sub-µm precision without the consumption of carrier fluid. The absence of nonspecific magnetic field induced bead aggregation makes NLM well suited for LOC applications based on MBA assays.The dependence of the critical frequency on the bead properties makes it possible to simultaneously separate bead populations due to differences in size, magnetization, or aggregation state, a technique we refer to as nonlinear magnetophoresis (NLM)UL27 gene of herpes simplex virus (HSV) 1 using single particle tracking on a MMA using reflectometry16. We have evaluated four different shapes of micromagnets for SPM bead transport and separation, i.e., circular (C), square (S), triangular (T), and rectangular (R) micromagnets. The shapes and the overall configuration of a MMA tested in this study have been selected having in mind the capabilities of the photolithographic lift-off process used for fabricating the MMAs. It is understood that other shapes of micromagnets may be envisaged, in accordance with the limitations of the fabrication method of choice. The results described herein can be nonetheless generalized and taken into consideration for the design of micromagnets having a shape other than those described in this work. The transport behavior of SPM beads on the MMAs have been linked to the micromagnets' lateral dimension and shape, which define the spatial distribution of the local magnetic flux density. We also demonstrate that the local magnetic flux density increases with the overall size of the micromagnets. These factors are linked to the observed critical frequency of the beads on the different arrays and importantly to the transport behavior of single beads and bead aggregates. We apply these MMAs to NLM separation of MBA assay products for the detection of a model protein biomarker and single-stranded DNA (ssDNA), as illustrated in Fig. 37. A direct, rapid and highly sensitive alternative to PCR methods, such as, single particle tracking using MMA, could allow NAT to be easily applied at emerging applications, i.e., next-generation sequencing and epigenetics, as well as POCT. The results presented here provide useful guidelines for the design of NLM separation systems with the aim of improving on-chip detection.This article builds on the recently published direct detection of < 200 copies of the ωc), immobilization frequency (ωi), relative velocities of the beads at ω > ωc, and their trajectories across an MMA. For the first criterion, a high ωc is desirable as it allowed rapid separation to be achieved, which reduces the overall processing time of the assay. The second criterion, the ωi is related to the separation efficiency of the NLM technique for different beads assemblies. For operational purposes the immobilization frequency has been defined as the frequency at which the average bead velocity decreased to less than 10% of its maximum value18. It has been observed that large differences in ωc and ωi correspond to a loss in separation efficiency due to the imprecise control of the frequency at which the single beads and dimers are separated18. The third criterion is related to the relative velocity of the different bead assemblies at ω > ωc. NLM separation efficacy is optimized when this difference is maximized. Finally, efficient separation results from controlled transport behavior but shifts in the orientation of the beads were observed from one row of micromagnets to another in several of the geometries.Figure ωc the velocities started decreasing until ωi was reached. The SPM beads and dimers had significantly different transport properties at ω > ωc on the four MMAs. The dimers showed a lower ωc than single SPM beads on C, S and T-micromagnets, whereas they had a critical frequency similar to the single beads on R. T-micromagnets had the lowest critical frequencies, i.e., 21.5 Hz for single beads and 16.5 Hz for dimers. R-micromagnets showed the highest critical frequencies, i.e., 31.0 Hz for single beads and 30.5 Hz for dimers. C and S-micromagnets showed an intermediate value of critical frequency for single beads, i.e., 27.0 Hz, and the dimer critical frequency decreased to 22.5 Hz and 23.5 Hz, respectively. The difference in the critical frequencies of the beads in the four arrays is attributed to the difference in the local magnetic flux density generated by the four micromagnet shapes, which is discussed in detail below. The variations in the lateral dimension of the micromagnets, and consequently in their volume, led to lower (or higher) magnetic force and therefore to lower (or higher) critical frequency.Figure ωi of 25 Hz for T, 30 Hz for C and S, and 40 Hz for R-micromagnets, whereas the ωi of single beads was 25 Hz for T, 40 Hz for C and S, and 60 Hz for R-micromagnets. To facilitate the analysis of the sharpness of the transition from “mobile” to “immobile” behavior we introduce the parameter Ω = ωc/ωi. The parameter Ω for single beads was 0.61 for T, 0.67 for C and S, and 0.51 for R-micromagnets. The parameter Ω for the dimers was 0.66 for T, 0.75 and 0.78 for C and S, respectively, and 0.76 for R-micromagnets. The higher values of Ω for dimers (with respect to single beads) is associated with the tendency of the dimers to change their orientation of motion when the driving frequency increased to values higher than ωc, i.e., it was found that the longitudinal axis of the dimers (defined as the axis between the two SPM beads) rotated from normal to parallel to the direction of motion at high ω. This led to a reduction of the total magnetic force experienced by the dimers and thus to a reduction in the immobilization frequency. Finally, the bead motion was observed to be stable across each of the MMAs except for R-micromagnets, where a shift of the beads’ trajectory was occasionally observed in the y-direction, leading to variations in their apparent x-direction velocities.Significant differences were also observed in the immobilization frequencies of SPM beads and aggregates on the four MMAs, as observed in Fig. 26. Figure θxz = 0°, label 2 θxz = − 90°, label 3 θxz = − 180° and label 4 θxz = − 270°). The SPM beads and dimers have been drawn to scale on Fig. ωc,C is 87% of ωc,R for the single beads. The magnitude of the change in the magnetic flux density was also observed to be significantly lower along the outer edges for C-micromagnets, which suggests that the ωc for the dimers should be lower than single beads. Again, this was consistent with the experimentally determined transport properties of the dimers, i.e., ωc,C is 77% of ωc,R for the dimers.The transport properties of single beads and dimers were closely related to the magnetic flux density on the micromagnetic array and to their lateral dimensions. Previous studies have revealed that SPM beads traveling on C-micromagnets move to the point of maximum flux density created by the micromagnet and external magnetic field, as shown in Fig. 2θxz = 0°, label 2 θxz = − 90°, label 3 θxz = − 180° and label 4 θxz = − 270°). The magnetic flux density profiles experienced by single beads and dimers on S and T are presented in Figure ωc for the single beads and dimers on T, i.e., ωc,T is 80% of ωc,S for the single beads and ωc,T is 68% of ωc,S for the dimers. Two other trends are clear from Figure θxz = 0 and − 180° but was significantly larger for θxz = − 90 and − 270°. The change in the magnitude of the magnetic flux density was also observed to be significantly smaller along the outer edges on the S-micromagnets. This suggests that the ωc for the dimers should be lower than single beads, which was consistent with the observed transport properties. Second, the shape of the magnetic flux density profiles for T are not symmetric. This suggests that these arrays will have asymmetric NLM separation behavior based on the x-direction of motion of the SPM beads.Figure ω < ωc. The lateral dimension of the dimers exceeded the lateral dimension of the C, S and T-micromagnets. Thus, the dimer experienced a lower local magnetic potential energy on these MMAs compared to single beads, resulting in lower critical frequencies30. In the case of R-micromagnets, the magnetic flux density experienced by the dimers was comparable to that experienced by the single beads due to the shape of the magnetic flux density maxima. The results highlighted the sensitivity of the NLM technique to the local magnetic flux density and confirmed the versatility of the MMA design, which obviously can be adjusted to meet specific experimental requirements associated with SPM beads and their aggregate properties. Based on the criteria previously defined it was concluded that for the applications of interest of this work the C and S-micromagnets were the most suitable designs, i.e., these two shapes gave the highest critical frequency combined with the minimum overlap between the velocity profiles of the single bead and dimer populations. R was less desirable due to the unpredictable transport behavior of the SPM beads and limited capacity to distinguish between single beads and dimers.The NLM microscope experiments allowed us to characterize the transport properties of different MMAs, in terms of critical frequency, stability of the motion, and separation capabilities. The magnetic flux density on all four MMAs forced the dimers into an orientation in which they traveled with their longitudinal axis orthogonal to the direction of motion for UL27 gene of HSV-1. In the MBA assay, the SPM beads were first incubated with the analyte for approximately 5 min and then a magnetic field was used to induce rapid bead-bead reaction and separation . In previous publications on bead-based assays related to GMR detection38, or to fluidic force discrimination39, the sub-linearity of the dose–response curves at high target concentration was attributed to magnetic and steric hindrance effects. At low target concentrations, the formation of SPM bead dimers is statistically favored over the formation of larger bead clusters21. At higher target concentrations each SPM bead captures multiple target molecules, and the formation of bead aggregates formed by multiple SPM beads tends to occur (confirmed by microscopy observation), a phenomenon that may decrease the efficiency of the assay due to the lower binding capabilities of bead clusters40. SPM beads with smaller size  could be used to improve the sensitivity of the assay, however the characteristic dimensions of the micromagnet arrays would need to be adjusted accordingly, in order to maintain efficient transport and separation capabilities: the lateral dimension and the gap between adjacent micromagnets should be reduced for achieving smooth transport of such small beads.The dose–response curves for the MBA These NLM results are in qualitative agreement with the flow cytometry measurements presented in Fig. ωc and ωi, was observed to increase in frequency with the increase in the maximum magnetic flux density, e.g., see Fig. ωc and ωi, as shown in Fig. ωc’s and ωi’s. However, these MMAs were able to separate monomers from dimers due to the significant difference in the magnetic flux densities at the edges of the micromagnet, which disproportionally influenced the transport properties of the dimers. These results suggest that high-efficiency NLM separation will be achieved with micromagnets that have lateral dimensions that are larger than monomers but smaller than dimers. This conclusion is subject to the observation that dimers can change their orientation at high NLM frequencies to reduce the hydrodynamic force.In this article, we examined the NLM transport of SPM beads based on different shape and size micromagnets. It was found that the transport behavior of both the SPM bead monomers and dimer was closely related to magnetic flux density produced by the micromagnet array. For the monomers, the transition from linear to nonlinear transport, i.e., the magnitude of 5 single beads and dimers. The lower aggregation level measured with NLM appears to result from the adhesion of the SPM dimers and larger aggregates to the silicon dioxide MMA surface, resulting in decreases in the apparent number of aggregates. We anticipate that the sensitivity of the NLM MBA assays will be equivalent to the flow cytometer once the nonspecific interactions of the SPM beads has been mitigated using a passivating surface chemistry. NLM also allowed us to work with very small number of bead aggregates, and thus molecules, which could have significant advantages for next-generation sequencing and epigenetic analyses where it is desirable to avoid PCR amplification.An S-chip was used to separate and detect two different biological targets to evaluate the efficiency of the NLM separation process and detection of dimers for the MBA assay using a simple optical microscope. Our previous studies have demonstrated that sensitivity of this technique is highly dependent on the parameters used for the MBA assay, i.e., type of beads used, surface chemistry, reaction times and volumes. After the SPM beads are added to the MMA the desired biological targets were isolated from the sample in a few seconds after the rotating magnetic field was activated. The sensitivity of the NLM MBA assays was similar to those obtained from a flow cytometer, with both techniques analyzing as many as 10A microscope was used to detect the SPM bead aggregates in this study, which was useful for the characterization of the transport processes and had single particle sensitivity, but obviously is not easily automated. Advances in optical and magnetic sensor design should allow detectors to be integrated into MMAs and the resulting LOC device promised to allow rapid, high-sensitivity analysis of nucleic acids, proteins, viruses or cells to be performed from complex media with minimal consumption of fluids.31. Briefly, the applied magnetic field rotating in the xz-plane was generated by three iron core electromagnets supplied with sinusoidal signals with a 90° phase difference via a two-channel function generator . This signal was amplified to the desired current using two programmable amplifiers . An ellipsoidal rotating field, with components of 30 G in the x-direction and 35 G in the z-direction, was used consistently in all the experiments. The transport of the SPM beads in the device was characterized with an Axioskop2 optical epi-illumination microscope .The NLM system used in this study has been described in previous publications2 kg−1 and density of 1.6 × 103 kg/m3. The bead aggregates were formed by BBSA at a concentration 10–9 mol dm−3. The experimental data reported in this paper refer to single beads and to aggregates formed by two beads, which was the most statistically favored shape in the samples at this BBSA concentration. The rare clumps of beads were not considered in these measurements since their behavior was not predictable, mainly due to an out-of-plane rotation during their in-plane motion across the MMA. The rotational frequency of the magnetic field was varied from 1 Hz up to 60 Hz, with increasing frequency steps. The distance travelled by the beads over a defined number of microscope frames was measured manually in the Zeiss AxioVision software. On average 15 beads or aggregates were analysed for each data point presented. In the high frequency regime, the SPM beads rapidly oscillated without moving from one micromagnet to the next one. In this regime the beads were classified as “immobile”. The ωc of the beads was defined as the frequency at which the average bead velocity started to diverge from the velocity of the translating potential energy landscape, ωd/2π, and was estimated based on the bead frequency response curves.The MMAs presented in Fig. x- and y-direction was 8 μm for circular, triangular and square micromagnets, whereas it was 8 μm in the x-direction and 16 μm in the y-direction for rectangular micromagnets.The micromagnet fabrication followed a standard photolithographic lift-off process. The micromagnets were fabricated on a silicon wafer via electron-beam deposition using 10 nm chromium, 100 nm cobalt, and 10 nm chromium. The bottom layer of chromium was used to enhance the adhesion of the micromagnets to the silicon substrate, whereas the top layer of chromium protects the cobalt from oxidation. A 600 nm thick spin-on-glass  layer was added on-top of the micromagnets to create a planar surface and ensure further protective layer for the cobalt. After the fabrication process the micromagnets were magnetized along the positive x-direction with a 11 kg impulse magnetizer . Four different shaped micromagnets were fabricated, i.e., circular , pH 7.4, with 0.1% Tween (PBST). The assay was performed by adding streptavidin SPM beads to 300 μl of the BBSA solutions to achieve a final bead concentration of 107 mL−1. The solution was incubated at room temperature for 5 min and then subjected to magnetic field activated aggregation, i.e., the sample was placed in a 2.5 kg magnetic field for ~ 1 min at which time the majority of the beads came out of solution. The tube was then rotated 90° in the magnetic field to allow the SPM beads to roll over each other and react. Approximately 100 μl samples of the product of 10−7, 10−9, 10−11 and 10–13 mol L−1 BBSA reactions were added to a S-micromagnet MMA for separation and detection with an optical microscope.Figure –7, 10–9, 10–11 and 10–13 mol dm-3 of TPT buffer . Streptavidin beads were added to the solution containing the target dsDNA, and the mixture was incubated at room temperature for 30 min. After this step, the dsDNA coated beads were washed with TPT buffer and reacted with biotin (0.5%) in 1 mL of TPT buffer for 30 min. After washing with TPT buffer, the beads were incubated with antidigoxigenin functionalized SPM beads for 30 min on a rotating wheel. The sample containing SPM beads was placed in a magnetic field of 2.5 kg until most of the beads came out of solution. Once the SPMs were collected the tube was rotated by 90° in the magnetic field. Approximately 100 µl samples of the product were added to a S-micromagnet MMA for separation and detection with an optical microscope.Figure Beads aggregation were also evaluated using a flow cytometer  with two light scattering detectors and four fluorescence detectors. The percentage of monomers and aggregates in the sample were determined by gating individual areas of the scatter plots with the foreword scattering area (FSC-A) against the foreword scattering height (FSC-H). Individual monomers have lower FSC-A and FSC-H values, and aggregated beads are easily identified and discriminated from monomers due to the higher FSC-A and FSC-H values. Each assay was run in triplicate and the average values are plotted in all graphs.Supplementary Information.


The current outbreak of the coronavirus disease‐19 (COVID‐19) pandemic worldwide has caused millions of fatalities and imposed a severe impact on our daily lives. Thus, the global healthcare system urgently calls for rapid, affordable, and reliable detection toolkits. Although the gold‐standard nucleic acid amplification tests have been widely accepted and utilized, they are time‐consuming and labor‐intensive, which exceedingly hinder the mass detection in low‐income populations, especially in developing countries. Recently, due to the blooming development of photonics, various optical chips have been developed to detect single viruses with the advantages of fast, label‐free, affordable, and point of care deployment. Herein, optical approaches especially in three perspectives, e.g., flow‐free optical methods, optofluidics, and surface‐modification‐assisted approaches, are summarized. The future development of on‐chip optical‐detection methods in the wave of emerging new ideas in nanophotonics is also briefly discussed. © 2021 WILEY‐VCH GmbHOptical methods for virus detection have the advantages of fast, label‐free, affordable, and point of care deployment. Herein, optical approaches in three perspectives, e.g., flow‐free optical methods, optofluidics, and surface‐modification‐assisted approaches, are summarized. The future development of on‐chip optical‐detection methods in the wave of emerging new ideas in nanophotonics is also briefly discussed. In tropical countries such as Singapore, the mosquito breeding also leads to the spread of dengue disease, leading to 4.2 million cases worldwide in 2019.Outbreaks of emerging infections occur more frequently and have more severe global impact in recent decades due to continuing population growth and urbanization.1, 2 The public health measures in global travel restrictions, social and economic activities limitations affect more than half of world populations. Such measures had a huge global impact and caused severe economy recession. For instance, USA experienced two consecutive quarters of declines in GDP in the second and third quarters of 2020. It even recorded its steepest quarterly drop in the second quarter of 2020. What is even worse is that the second wave of COVID‐19 has sent many European countries back into lockdown, with a continuous increased death toll.Up to the early October 2020, over 50 million people have been infected by SARS‐CoV‐2, and the mortality rate is 2.48%.9 The  The most widely used detection technique is the polymerase chain reaction (PCR), which shows a high specificity. However, the high testing rate requires lots of specially trained personnel and detection kits, which are costly. In addition, it takes hours to obtain the results, which is also time‐consuming. Most significantly, PCR test suffers from false‐positive cases due to sample cross‐contamination and the fact that the detection of viral nucleic acid does not definitely associate with infectivity. Therefore, protein tests such as those used to detect antibodies in patient samples are used as complementary tests to confirm the testing results.The nationwide or city‐wide coronavirus testing has been approved to be an efficient way to screen out the infectious entities from public, thus cutting off the source of transmission. Successful countries in doing so include Australia, Singapore, and China.15 The The noninvasive optical methods also preserve the viability of viruses, potentially applicable for the study of the transmissibility, virulence, evolvability, and immunology of virus. For instance, epitope variation could result in escape mutants that are selected under immune pressure to render a potential vaccine inefficacious. Also, optofluidic methods fuse the state‐of‐the‐art techniques in nanophotonics and microfluidics to enable a faster and real‐time detection with a low sample consumption.Optical methods for virus detection have been widely explored recently due to the advantages of fast, label‐free, reusable, affordable, and portable for point of care (POC) deployment.25 TheIn this review, we provide an overview of current on‐chip optical methods for the detection of various viruses, such as SARS, H1N1, MERS, Zika, and COVID‐19. This article focuses on three distinct aspects, i.e., flow‐free optical detection, optofluidic approaches, as well as surface‐modification‐assisted optical methods. We review the optical mechanisms of those methods and summarize critical characteristics such as limit of detection (LOD) and detection time. Future perspective of current cutting‐edge techniques in nanophotonics that might be implemented for virus detection is also discussed.2Figure  to trap virus into the nanocavity. When a virus is trapped, the resonance of the nanocavity will be slightly shifted, which can be detected by a spectrometer. Meanwhile, the trapped virus also allows multi‐parameter analysis of single virions, such as heterogeneity in transmission and infection. Viruses can also be quantified by observing the accumulated signals from multiple viruses trapped. The ring resonator is another alternative configuration that stores light in the whispering gallery mode (WGM) with a high quality factor (up to 106). However, the exerted optical force is much weaker than those in nanocavities, and incapable to directly trap single viruses. In such a case, one can either rely on the free diffusion of virus to the surface of the ring resonator or modify the surface with specific receptors to assist the immobilization of virus on the surface. The output signal will change when the effective refractive index of the medium is disturbed by the existence of viruses. The viruses can also be quantified by the shifting of accumulated signals. Apart from nanostructures, many other flow‐free optical methods are also well developed, for instance, surface plasmon resonance (SPR), surface enhanced Raman scattering (SERS), multimode interference (MMI), fluorescence staining, etc. These methods will be reviewed extensively in the following section.Many conventional flow‐free optical detecting methods originate from the fast development of structural design in silicon photonics, for instance, nanocavities and resonators as shown in Figure 32 to The synergy of nanophotonics and microfluidics to fuse advantages from both fields empowers many possibilities in optical detection and manipulation. The robust conveyer of viruses in the delicate microchannels boosts the detection speed and enables real‐time diagnosis. For instance, single viruses can be confined in the hydrodynamic focusing of the flow cytometry as shown in Figure Table Other optics‐assisted methods include surface modification (SM). For instance, the sandwiched molecule structures are used to enhance the detecting signal and increase LOD as shown in Figure −1. Strategies such as fluorescence labeling can be adopted to enhance the signal from the high‐speed flow stream. Apart from those methods, some biomedicine‐dominated techniques also use optics as auxiliary tools to help to detect viruses, such as on‐chip PCR and SMs. Those methods seek for the interactions of biomolecules and use optical techniques to boost the detection speed and push down the LOD of conventional methods, such as PCR and enzyme‐linked immunosorbent assay (ELISA).The flow‐free optical‐detection methods focus on the utilization of advances in photonics to probe the signal variations caused by the different optical properties of viruses. The volume of detection is normally quite small, e.g., in nanoliter or picoliter. The detection LOD depends on the optical approach itself when no signal amplification strategy from the virus modification is implemented. The optofluidic methods fuse the microfluidic and optical systems to transport viruses to the detection region. These methods are requested to have capabilities of fast and sensitive signal outputs, correlating with a relatively high flow rate up to μL min3 SPR sensors have manifested themselves as versatile and almost the most widely used biochemical sensors due to the great characteristics in sensitivity, biocompatibility, easy implementation, etc. The principle of SPR sensor relies on the propagation of excited surface plasmons along the interface of a thin metal  and a dielectric (aqueous medium for virus detection) layers by the total internal reflection of the incident wave as shown in Figure  The surface of the metal layer can also be modified by coating with different antibodies targeting on specific viruses to immobilize them onto the surface. Each bound compound such as ligand and virus on the surface will shift the output signal, enabling the measurements of the binding affinity and virus quantity. To date, many SPR‐based toolkits or commercial systems have been developed to detect different viruses, such as Ebola, H5N1, infectious bursal disease virus (IBDV), hepatitis B virus (HBV), COVID‐19, etc. Detailed information of SPR sensors can be found in many other comprehensive reviews focused on this specific topic. Another classical sensor is based on the WGM of the ring resonator or microspheres as shown in Figure  Vollmer et al. reported the detection of the size and mass (5.2 × 10−16 g) of an Influenza A virus from the shift of resonance. He et al. developed a microlaser using WGM to detect individual polystyrene nanoparticles with 15 nm in radius, gold nanoparticles (10 nm), and influenza A virions in air, as well as polystyrene nanoparticles (30 nm) in water. With the naturalization of nanofabrication techniques, the WGM‐based sensors are now becoming more compact and portable for rapid POC diagnosis. This technique for virus detection relies on the virus diffusion onto the surface, which hurdles the LOD in aqueous medium because most viruses might not be bound. This limitation can be overcome by the assistance of SM with antibodies, which, however, requires specially trained personnel and compromises the detection time. An integrated optical Young interferometer sensor was proposed by Ymeti et al. for the direct, ultrasensitive, and real‐time detection of herpes simplex virus type 1 (HSV‐1) as shown in Figure  The detection limit is quite promising at a concentration of 850 particles per mL in serum. Different from the microring resonators or microsphere cavities, the Young interferometer sensor requires a charge‐coupled device (CCD) camera instead of a spectrometer, and is capable of an extremely high sensitivity, short response time, and multiplexing simultaneously. It is anticipated to be a strong candidate for a POC viral diagnostic kit. A MMI waveguide can generate wavelength‐dependent spot patterns within the entire visible spectrum to facilitate the multiplexed detection of single biomolecules as shown in Figure  The mass of individual viruses can be detected using silicon cantilever beams with a nanoscale thickness as shown in Figure  Based on the change in resonant frequency with the relation of virus particle mass, single vaccinia virus particles were measured with an average mass of 9.5 fg. However, this method measures only the dry mass of viruses, whereas virus study is commonly performed in aqueous medium. The sizes of viruses could be extracted and compared using the atomic force microscopy (AFM) to distinguish different virion species as shown in Figure  Other mechanisms such as capsid–genome interactions, morphological changes that drive viral uncoating and maturation can also be unveiled by the AFM.Following the pioneer works on gas detection and biosensing in 1980s,38 SPR from the strong resonance of cavities in photonic crystals trap single viruses easily from the aqueous medium. Optical trapping has the advantage of noninvasive and is capable to study the HIN1 influenza virus and measure the stoichiometry of antibody‐binding interactions using near‐field light‐scattering technique. It was reported that an H1N1 influenza virus was approximately bound with 26 ± 4 anti‐influenza antibodies as shown in Figure  More detailed discussion about the advances in optical manipulation of biomolecules can be found in recently published review articles. Metallic nanoparticles such as gold nanosphere  The shift of the resonance frequency in the transmission or reflection spectrum; 2) The characteristic absorption in the spectrum. The most classical strategy is taking the advantage of the dielectric constant or refractive index of viruses that can shift the transmission or reflection peak or dip in the spectrum. Park et al. reported a highly sensitive virus‐detection method using traditional split‐ring resonators with various capacitive gaps in the terahertz regime . The LC emulsion methods were shown to detect the virus with a low concentration (down to 104 pfu mL−1). To detect the early stage of virus affection in live cells with a high resolution and high speed, Huang et al. reported a coherent bright‐field microscope that provides the spatiotemporal resolution  in three dimensions and microsecond temporal resolution (up to 100 000 frames per second). This unprecedented performance offers many possibilities to elucidate the virus–host interaction, which is essential for the diagnostics, vaccine, and antiviral drug developments.Based on the emulsion of monodispersed liquid crystal (LC), Sivakumar et al. developed a versatile sensing method to detect both enveloped and nonenveloped viruses Figure .98]98[9n Figure .[99]4Figure  This chip can be performed in a flow rate of 1.2 μL h−1. By implementing optical tweezers in a microchannel, single viruses can be trapped and analyzed. Pang et al. reported that the trapping of individual HIV‐1 viruses allowed the multi‐parameter analysis of single virions in culture fluid environmentally mimicking native conditions  and small‐enveloped‐RNA viruses (pseudotyped Ebola and vesicular stomatitis virus) were detected with ultra low noise signal ratio and a high dynamic range in three orders.Optofluidics fuses two state‐of‐the‐art research fields, nanophotonics and microfluidics, thereby exhibits unparallel advantages from each technique to realize novel and powerful functionalities. It is a paradigm to transport single bioparticles such as viruses to the detection area in the microchannel and use various optical approaches to detect them. The assistance of fluid boosts the detection speed and enables real‐time diagnosis of viruses. Developing high‐sensitive and fast optofluidic detection device is in urgent demand nowadays due to frequent outbreaks of pandemics worldwide in the past two decades. A good example is the development of an optofluidic device for rapid and label‐free detection of biomarkers using gold‐coating nanopillar enabled SERS Figure 5a.[100s Figure .[34, s Figure .[102Figure  The heterodyne detection eliminated phase variations from trajectories of different particles to improve the recognition accuracy, showing great potential in the screening and detection of different viruses . Later, Mitra et al. combined the heterodyne interferometry with dark‐field microscopy for HIV virus and bacteriophage detection  in clinically relevant media. To reduce the sample preparation time and reduce the potential contamination, Cai et al. proposed an optofluidic chip that could implement sample preparation and target preconcentration on a polydimethylsiloxane‐based optofluidic chip , providing a virus specificity of 90%. It was also capable of sequencing and could be used for timely monitoring of viral outbreaks. To improve the limit of measurement speed by the fluorescence emission lifetime and duration by the photobleaching and thermal diffusion, Faez et al. used a hollow optical fiber containing the nanofluids to track 20 nm nonstained dielectric particles as well as individual cowpea chlorotic mottle virus virions with size of 26 nm at a rate of over 3 kHz for a duration of tens of seconds  cantilever‐actuated system could be used for detecting Hepatitis A and C viruses with dynamic range over 1000 and a limit of detectable concentration at 0.1 ng mL−1 or 1.66 pM , a pathogen of great agricultural importance, was detected by silicon photonic micro‐ring resonator with an LOD of 10 ng mLn Figure 120 To Before initiating the assay, the sensor surfaces were functionalized with a self‐assembled monolayer of 3,3′‐dithiobis (sulfosuccinimidyl propionate) to generate the thiol–gold linker, and subsequently bound with a layer of protein A/G to serve antibody anchors. Layers of self‐assembled monolayer plus protein A/G had a thickness around 6.1 nm to prevent the loss of irradiative fluorescence signals. Figure −1, which is 240 000‐fold higher in sensitivity compared with the existing rapid EBOV immunoassay (53 ng mL−1). Another work also introduced N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride and N‐hydroxysuccinimide to activate the modified SPR surface for EBOV detection.Using LSPR, gold was used to form nanodisks and then plasmonic nanocavity array was used to detect Ebola virus antigen (EBOV) with a sandwich assay Figure .[128 Figure In other works, optofluidic systems with MMI multiplexing were used to detect Zika nucleic acid and protein biomarkers.130 FiAlthough there are various surface treatment methods, the common principle is to create a biorecognition layer on the chip surface through a few chemical or biological modifications. First is the surface activation with active group. Then single or a few linkers is introduced following the active group. Finally, the capture probe which has specificity determination to the target is linked.5.2 Despite of good performance of conventional RT‐PCR, it also has some drawbacks such as long reaction time (≈2 h) and undesirable sensitivity , which have been evidenced in the battle against COVID‐19.In COVID‐19 pandemic, RT‐qPCR, a version of NAAR, becomes a preferred method of coronavirus test that contributes in alleviating the spread of coronavirus.131 De Au nanofilm   ,134 Au) Figure , photoni) Figure  as an ex and virus residue on objective surface, and thus hinders its application in clinical practice. Digital PCR (dPCR) could be helpful to overcome this problem. Through distributing targets into isolated microreactions with nanoliter or picoliter volume, dPCR can realize single nucleic acid detection after amplification but suffers from long reaction time. Considering the short reaction time of photonic PCR, some groups have done preliminary work in exploring photonic digital NAAR to achieve ultrafast and ultrasensitive infective pathogens detection. Photonic digital NAAR with the merits of high sensitivity and short reaction time show bright prospective in POC diagnosis and could be a powerful technology in response to the next virus outbreak.Although the photonic PCR dramatically shortens detection time, its sensitivity cannot meet the requirement of some clinical scenarios; for instance, the detection of virus in the early stage of infection132 an6−1, but the prices of machine and each chip cost approximately hundreds of thousands of US dollars and a few hundred US dollars, respectively. Moreover, the reagent solutions may also cost hundreds to thousands of US dollars. The nanoplasmonic platform may serve as a paradigm for the virus detection with the assistance of antibody either in a single layer or at a sandwiched molecule structure. The cost is also acceptable due to the standard sample fabrication and surface treatment processes. Another fast, sensitive, and promising approach is the photonic digital NAAR, which detects high‐heterogeneity virions with affordable expenses, suitable for conducting experiments in almost all virion‐accessible hospitals and labs.Clinical utilities of on‐chip optical methods need to assess many critical factors, such as speed, volume, cost, LOD, heterogeneity, etc. Optical methods are fast with detection time from milliseconds to several hours. However, most flow‐free detection methods can only detect samples of small volumes, normally in picoliter or nanoliter. Meanwhile, many viruses have similar optical properties , imposing great difficulties in the detection of virion heterogeneity using these methods. The commercial SPR machine can be operated at a flow rate of several μL min Yesilkoy et al. reported a metasurface to achieve an LOD of three molecules per μm2 by using a pixel‐based thresholding method. An imaging‐based nanophotonic device was proposed recently for the detection of molecular fingerprints using a 2D pixelated dielectric metasurface. Each pixel was tuned to a discrete frequency, and its amplitude would be affected by the presence of biomolecules. The affected spectrum was then translated into a barcode‐like absorption map. This cutting‐edge nanophotonic technique resolved absorption fingerprints without the requirement of spectrometry, being potentially applicable for detecting species of viruses. Park et al. reported the observation of exceptional point (EP) in a plasmonic system by hybridizing detuned resonances in multilayered plasmonic structures. The EP system pushed up the detection sensitivity of anti‐immunoglobulin G to 4821 nm per RIU and stipulated an LOD of 15 × 10−12 g L−1, which outperformed previous plasmonic array sensors. High‐order EP systems were also found to enhance the sensitivity. Meanwhile, Jing et al. used the enhanced mode splitting and a strongly modified transmission spectrum to enhance the sensitivity of the WGM sensor. Their device did not require complex materials, hybrid systems, optical gain, or low temperatures. Those progresses in extreme photonic systems will enable future advances in faster and more sensitive biosensing.Recent advances in nanophotonics have made huge progress in the sensing of biomolecules. All‐dielectric asymmetric metasurfaces with the intriguing physics of bound states in the continuum, have unprecedented feature of high‐quality resonances, which provide an ultrasensitive label‐free analysis platform for biosensing.155 YeComparing with gold‐standard nucleic acid assays using PCR testing and typical ELISA, optical methods empower a faster, label‐free and single‐virus detection, providing alternative solutions to tackle current outbreak of pandemics. More opportunities in sequencing, genomic and heterogeneity analysis, viral pathogenicity, which are not attainable by conventional PCR, can also be studied using optical chips. Meanwhile, on‐chip PCR is showing more and more evidence in the improvement of detection sensitivity. Although there is still a gap between the massive commercialization and laboratory study, future simplification and increased specificity plus the intrinsic high sensitivity of photonic devices would contribute hugely to the affordable nation‐wide detection to prevent and stop pandemics. The precise control of micro‐environment conditions for drug testing in the optofluidic chip will improve the efficiency in vaccine and antiviral drug development. All these possibilities help to accelerate the development of virus diagnostics, vaccines, and antiviral drugs to tackle unknown pandemic outbreaks such as COVID‐19.The authors declare no conflict of interest.


In China, the disease burden of cervical cancer remains substantial. Human papillomavirus (HPV) vaccines are expensive and not yet centrally funded. To inform immunization policy, understanding the economic burden of the disease is necessary. This study adopted a societal perspective and investigated costs and quality of life changes associated with cervical cancer from diagnosis to one year after final discharge in Henan province, China.Inpatient records of cervical cancer patients admitted to the largest cancer hospital in Henan province between Jan. 2017 and Dec. 2018 were extracted. A telephone interview with four modules was conducted in Jun.-Jul. 2019 with a 40% random draw of patients to obtain direct non-medical costs and indirect costs associated with inpatients, costs associated with outpatient visits, and changes in quality of life status using the EQ-5D-5L instrument. Direct medical expenditures were converted to opportunity costs of care using cost-to-charge ratios obtained from hospital financial reports. For each clinical stage (IA-IV), total costs per case from diagnosis to one year after final discharge were extrapolated based on inpatient records, responses to the telephone interview, and recommendation on outpatient follow-ups by Chinese cervical cancer treatment guidelines. Loss in quality-adjusted life years was obtained using the ‘under the curve’ method and regression predictions.A total of 3,506 inpatient records from 1,323 patients were obtained. Among 541 randomly selected patients, 309 completed at least one module of the telephone interview. The average total costs per case associated with cervical cancer from diagnosis to one year after final discharge ranged from $8,066-$22,888 (in 2018 US Dollar) and the quality-adjusted life years loss varied from 0.05–0.26 for IA-IV patients.The economic burden associated with cervical cancer is substantial in Henan province. Our study provided important baseline information for cost-effectiveness analysis of HPV immunization program in China. The disease burden of cervical cancer has been substantial globally, especially for less developed regions. In 2018, there were approximately 570,000 new cases and 311,000 cervical cancer related deaths, more than 84% of which were from low- and middle-income countries (LMIC) [Persistent human papillomavirus (HPV) infection is a necessary cause of cervical cancer . If leftHowever, HPV vaccines are not currently centrally funded in China–individuals have to pay the full amount of the shots out-of-pocket. The total costs of three doses of HPV vaccines are $140-$444 , corresponding to around 3.3% to 10.4% of the annual disposable income per capita in China as of 2018, which is very expensive for most to afford. To support the decision of introducing HPV vaccines into China’s National Immunization Programme, evidence based on cost-effectiveness analyses is necessary. Estimates of economic burden of cervical cancer in China provide important baseline data for such analyses.Following up cervical cancer patients prospectively over the entire treatment process since diagnosis is an ideal study design. However, it is hard to implement due to the long treatment duration of cancers. Previous studies that examined economic costs associated with cervical cancer in China mainly adopted a hospital-based design and focused solely on total medical expenditures of inpatient stays –9. Shi eIn this study, we adopted a societal perspective, extracted inpatient records, and conducted a retrospective telephone survey among cervical cancer patients who attended the largest cancer hospital in Henan province to estimate the economic burden of cervical cancer. We considered both direct and indirect costs from diagnosis to one year after final discharge. We also assessed the quality of life impact of cervical cancer using QALY loss.th out of all 31 provinces in 2017. During 2011–2015, the central region of China accounted for 31% of total hospitalization expenditures for tumor treatment across the country, and specialized cancer hospitals amounted to 50% of the total [Henan province is located in central China, with a per-capita GDP level ranking the 18Patients who were admitted to the Gynecology Department at Henan Cancer Hospital between January 1, 2017 and December 31, 2018 were considered the potential participants of this study. Then, patients who completed inpatient treatment and were recommended for regular outpatient follow-ups were identified based on discharge summaries. Information on the entire treatment process of the patients was obtained based on treatment history on their last observed inpatient records.Finally, a 40% random sample without replacement of all patients with inpatient records were invited to a telephone survey, where information on socioeconomic characteristics (education level and monthly family income) were collected. Patients were asked to recall costs associated with cervical cancer from diagnosis to the interview day and changes in quality of life states.The telephone survey was conducted between June 3, 2019 and July 25, 2019 by LinkDoc, a company specializes in patient follow-ups in China. Staffs from LinkDoc were trained and supervised by the research investigators. A computer-assisted telephone interviewing system was used to record answers directly into a computer. A patient was classified as unreachable if we failed to get in touch with her after four attempts made on different days. After verbal consents from the participants, demographic and clinical information was verified using their most recent inpatient records.There were four modules in the survey. Accordingly, four samples were generated based on complete responses to each module. First, the EQ-5D-5L instrument was used to assess the health states of the patients. EQ-5D is a standardized five-dimensional preference-based health-related quality of life questionnaire designed by the EuroQol Group, which consists of a descriptive system with five dimensions and a visual analogue scale (VAS). The instrument has been validated in China and among cervical cancer patients in Asian populations , 17. PatSecond, non-medical costs associated with inpatient episodes were collected. Average direct non-medical costs  and indirect costs (income losses incurred to the patients and caregivers) associated with each inpatient episode were obtained. For respondents who could not provide a detailed value for income loss, changes in employment status and loss of work days associated with inpatient episodes were asked and used as proxies.Third, average direct medical expenditures (including both out-of-pocket and reimbursable portions) associated with each outpatient visit were obtained. Fourth, average direct non-medical costs associated with each outpatient visit were acquired. Average indirect costs associated with outpatient visits were not collected because most participants thought the amount was trivial. Indirect costs due to productivity loss and premature death were not considered.The right half of From the responses provided by patients who completed the inpatient module during the telephone survey, costs per inpatient episode were calculated by adding up the direct medical costs, the direct non-medical costs, and the indirect costs. Generalized linear regression models (GLM) were used to identify predictors of costs. Demographic, socioeconomic, and clinical characteristics and treatment methods were investigated.Then, total costs per case by clinical stages from diagnosis to one year after final discharge were extrapolated. The treatment processes were broadly divided into two phases: the inpatient phase  and the outpatient phase .For the inpatient phase, direct medical costs were calculated based on two parameters: first, the average direct medical costs associated with inpatient episodes, which were estimated by treatment type using all eligible inpatient records; and second, the average number of hospitalization, which was estimated by treatment type received during the entire inpatient phase using only records of patients who experienced final discharge. Using the group of patients with IA cervical cancer as an example: there were 37 patients who experienced final discharge, 36 of whom were hospitalized once for surgery, and one patient for five times . Therefore, the average number of hospitalization with surgery and chemotherapy received per IA patient was 1 (37/37) and 0.11 (4/37), respectively. Direct medical costs were calculated by multiplying the aforementioned parameters for each treatment type and then adding up their products for each clinical stage. Direct non-medical costs and indirect costs were calculated by multiplying the average of the two costs associated with each inpatient episode with the average number of hospitalizations during the inpatient phase. If the respondents provided an un-useable answer to indirect costs, their corresponding estimates were derived using the human capital method based on national average income per capita and their losses of workdays.In the outpatient phase, the Guidelines for Diagnosis and Treatment of Cervical Cancer, issued by the Committee of Gynecological Oncology of the Chinese Anti-Cancer Association, recommends patients to be regularly followed-up as outpatients every two months in the first six-month period and every three months in the second six-month period after final discharge, totaling five outpatient visits . Thus, tTo examine the quality of life impact of cervical cancer, responses to the EQ-5D descriptive system for the current and worst health states were converted to health utilities using a China-specific value set . The basIn this study, the patient information was stored in coded encryption after data collection, and we had no access to information that could identify individual participants. We adjusted for inflation to match for 2018 US dollar value using a country-specific consumer price index and foreign exchange rates . All staThe study was reviewed and approved by the institutional review board at Henan Cancer Hospital  and Fudan University School of Public Health .From January 2017 to December 2018, there were a total of 4,169 inpatient admissions to the Gynecology Department at the study hospital. Of these, 3,506 records of inpatient episodes from 1,323 patients were eligible under the inclusion criteria. Among them, 844 patients had experienced final discharge and 541 (40%) randomly selected patients were invited to the telephone survey. Module completion rates varied from 26.1% (141/541) (the module on direct costs for outpatient visits) to 61.0% (330/541) (the EQ-5D module). Main reasons for non-completion included unwillingness to provide information, language barriers, and distrust of investigators.The distribution of inpatient phase duration among those who experienced final discharge in our study shows that patients with advanced cancers generally underwent more extended inpatient phases see . For IA For costs per inpatient episode, clinical stage and treatment methods were significantly associated with total costs, direct medical costs, and direct non-medical costs (col. (1)-(3) of The EQ-5D scores of the descriptive system and VAS suggest similar patterns. Compared to the norms of the Chinese population, cervical cancer patients had poorer health states . The aveThis study adopted a societal perspective and examined clinical stage specific costs and QALY loss associated with cervical cancer from diagnosis to one year after final discharge in Henan Province, China. Based on the inpatient records from the largest cancer hospital in the province and a retrospective telephone survey, we found that costs per case ranged from $8,066-$22,888 and QALY loss ranged from 0.05–0.26.To further understand the financial burden, we calculated the average time from diagnosis to one year after final discharge by clinical stages. We then divided total costs per case by these time estimates. The overall average was weighted by the proportion of each clinical stage admitted to the study hospital. We found that the average annual costs per cervical cancer patient were 1.7 times GDP per capita and 4.0 times per capita household disposable income in Henan Province as of 2018. According to a recent systematic review, patients in middle- and high-income countries only spend 75.52% and 30.12% of the local GDP per capita on cervical cancer treatment . Our resPrevious studies from China mainly focused on total direct medical expenditures during the inpatient phase  or the aAlthough previous studies that reported expenditures per inpatient episode were conducted in different areas of China –28, we fOur study contributed to the existing literature on the economic burden of cervical cancer in China due to a number of features. First, our estimates suffer less under-estimation because we accounted for a wide range of costs categories and considered the entire inpatient phase and a one-year outpatient phase. We highlighted the importance of indirect costs and costs during the outpatient phase as a component of financial burden, which makes up 9.4%-15.2% and 9.1%-29.0% of total costs . Second,This study speaks to the important policy concern on whether it is cost-effective for the central government of China to add the HPV vaccine into its national immunization program. Currently, HPV vaccines in the China market are produced by two foreign pharmaceuticals (GlaxoSmithKline and Merk & Co.) and injection costs are fully borne by individuals. Although the average annual costs per cervical patient were estimated to be 4.0 times per capita household disposable income in Henan Province, the costs of three doses of HPV vaccine are also expensive to afford (3.3%-10.4% of per capita household disposable income). In January 2020, a newly approved domestic bivalent HPV vaccine is being prepared for production. It is priced at nearly half of the costs of those provided by foreign drugmakers, which potentially increases the cost-effectiveness of the injection.Unfortunately, previous study on the economic burden of cervical cancer in China did not fully recognize the long and complicated treatment process of the disease, leading to an underestimation of the costs incurred to the whole society that HPV vaccines can prevent. Our study instead produced more accurate estimates for the treatment costs of cervical cancer and loss in quality-adjusted life years associated with the disease. These results provided crucial parameters for cost-effectiveness studies and are important for the decision of relevant immunization policy.Our study is subject to several limitations. First, information collected from the telephone surveys may be subject to recall bias. Information on non-medical and indirect costs may suffer measurement errors since it was obtained based on the memory of the participants. Second, the response rate of the telephone survey was not ideal which may potentially affect the accuracy of our results. Comparisons between patients in the final analysis and those not  Tables sS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S1 Questionnaires(ZIP)Click here for additional data file.S1 File(ZIP)Click here for additional data file.


Human adenoviruses (HAdVs) are common pathogens causing a variety of respiratory, ocular and gastrointestinal diseases. To accomplish their efficient replication, HAdVs take an advantage of viral small non-coding RNAs (sncRNAs), which have multiple roles during the virus lifecycle. Three of the best-characterized HAdV sncRNAs; VA RNA, mivaRNA and MLP-TSS-sRNA will be discussed in the present review. Even though VA RNA has been extensively characterized during the last 60 years, this multifunctional molecule continues to surprise us as more of its structural secrets unfold. Likely, the recent developments on mivaRNA and MLP-TSS-sRNA synthesis and function highlight the importance of these sncRNA in virus replication. Collectively, we will summarize the old and new knowledge about these three viral sncRNAs with focus on their synthesis, structure and functions. Human adenoviruses (HAdVs) are common pathogens causing a variety of gastrointestinal, respiratory, and ocular diseases in humans ,2. In adhttp://hadvwg.gmu.edu). However, most of the functional studies have been carried out using the genetically almost identical virus types 2 and 5 (HAdV-2 and HAdV-5) ..16].The last 60 years of research have expanded our understanding about the VA RNA regarding its synthesis, structure and function. However, there are still a number of interesting unanswered questions which need to be addressed. For example, a co-crystallization of VA RNAI and PKR is desirable to understand the details how VA RNAI binds to and inhibits PKR function. Moreover, since inflammation is such a central part of a virus infection, the recent finding that VA RNAI blocks NLRP3 inflammasome activation , urges fLimited data is available about the mivaRNA function during HAdV infection. Current knowledge about the VA RNA and mivaRNA functions are restricted to the studies based on HAdV-5 infections. Thus, it is important to expand the functional studies also to mivaRNAs expressed from other HAdV types, including more pathogenic and re-emerging HAdV-7 and HAdV-55 ,97. FurtThe discovery of the non-canonical MLP-TSS-sRNA also solicits for further studies. Do the other viral promoters also produce Ago2-associated TSS-sRNAs? Are the Ago2-associated TSS-sRNAs unique to a HAdV infection or do they also exist in other virus infections? It is known that mouse embryonic stem cells produce functional TSS-miRNAs . It willTaken together, we believe that the coming years will presents us with several exiting possibilities to explore the structural and functional details of the VA RNA, mivaRNA and TSS-sRNA, with an ultimate aim to apply this gained knowledge to design advanced HAdV vectors. These studies are especially important since modified adenoviruses have turned out to be suitable delivery vectors to vaccinate against SARS-CoV2 infections ,6.