Sumera Khizar

Amal A Al-Dossary

Nadia Zine

Nicole Jaffrezic-Renault

Abdelhamid Errachid

Abdelhamid Elaissari
email: abdelhamid.elaissari@univ-lyon1.fr

Contribution of magnetic particles in molecular diagnosis of human viruses

Keywords: Viruses, Magnetic nanoparticles, Extraction, Enrichment, Detection

Highlights

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References……………………………………………………………………………………….43

Introduction

Far-reaching pandemics of infections have been relatively rare all over history, but once they occur they can be alarming. These infections influence a large number of populations and cause millions of deaths every day. Even with the development of diagnosis and treatment by medicine, these epidemics are nature's mode of evoking us that despite our hard work; nature can never be controlled by us.

Viruses are one of the main sources of infection and global death, mostly due to the growth of impervious strains of viruses and the contrary side effects related to sustained usage that leads to reducing the significance of operative antiviral treatments [START_REF] Galdiero | Silver nanoparticles as potential antiviral agents[END_REF]. Humans have been combating these viruses since earlier our species had even progressed into its modern form. Thanks to immunization platforms, specific ailments that are caused to kill countless and gradually inactivate others have been eliminated. Still, certain lethal viruses around the world present a severe hazard to community health because of not having consistent sources to fight them. Inhibition of the spreading of the virus requires quick and accurate diagnosis and appropriate management of the contagious agents. Many specific diagnostic techniques are available for the extraction and detection of different deadly viruses (Table 1). In this situation, nanotechnology proposes an adaptable and reliable platform to detect diseases caused by viruses. Different carriers having nanoscale size (e.g., solid lipid nanoparticles, liposomes, inorganic nanoparticles, polymeric nanoparticles, and more) have appeared as a novel "antiviral agents" because of their exclusive chemical and physical characteristics [START_REF] Kim | Antimicrobial effects of silver nanoparticles, Nanomed[END_REF]. Additionally, the surface modification of nanoparticles widens their applications and unlocks their innovative potentials for adapting them to attain a future objective.

Table 1

List of some deadly viruses in human history and conventional systems used for their detection.

Type of virus

Average fatality rate Conventional detection system Ref Marburg virus (MARV) 23%-90%.

Polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) [START_REF] Marzi | Protection Against Marburg Virus Using a Recombinant VSV-Vaccine Depends on T and B Cell Activation[END_REF] Ebola virus (EBOV) 25%-90% Reverse transcription Polymerase chain reaction (PCR) [START_REF] Thi | Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates[END_REF][START_REF] Duan | Nanozyme-strip for rapid local diagnosis of Ebola[END_REF] Human immunodeficiency virus (HIV)

30%

Polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA [START_REF] Mamo | Emerging nanotechnology approaches for HIV/AIDS treatment and prevention[END_REF][START_REF] Lee | Development of a HIV-1 Virus Detection System Based on Nanotechnology[END_REF] Hantavirus (HPS) 36% Serology, reverse transcription Polymerase chain reaction (PCR [START_REF] Mattar | Diagnosis of hantavirus infection in humans[END_REF][START_REF] Kruger | Human pathogenic hantaviruses and prevention of infection[END_REF] Influenza virus(A, B, Spanish flu)

40%

Serology, rapid antigen testing, reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence assays, and rapid molecular assays. [START_REF] Li | Engineering influenza viral vectors[END_REF] Dengue virus 2%-20% Serological testing, and RNA amplification using reverse transcriptase PCR [START_REF] Ahmed | Climatic Conditions: Conventional and Nanotechnology-Based Methods for the Control of Mosquito Vectors Causing Human Health Issues[END_REF] Coronaviruses (SARS-CoV) -Reverse transcriptase PCR -Multidisciplinary investigations have been done for the progress of unconventional approaches to improve antiviral diagnostics and therapeutics, in which nanotechnologists play a vital role and shoulder their social accountability. As one of the game-changers of the previous era, amongst numerous science and technology fields, nanotechnology offers inventive ways to solve a broad range of difficulties and problems concerning inhibition, identification, and detection of viruses. Moreover, the efficiency of orthodox therapies is gradually vanishing away due to the increase in resistance against the virus, which could be positive because of the enhanced variation in peripheral protein sequence leading to new viral strain [START_REF] Kerry | Nano-based approach to combat emerging viral (NIPAH virus) infection, Nanomed[END_REF]. Furthermore, nanotechnology manipulates nanoparticles for viral extraction and detection generally, focused on most typical and fatal viruses, namely influenza, hepatitis, HIV, and ebola counting the ongoing novel coronavirus COVID-19 [START_REF] Yang | Application of Nanotechnology in the COVID-19 Pandemic[END_REF]. Through nanotechnology; viruses can be identified or deactivated by the labeling of viruses with nanoparticles, constraining the duplication of the virus on entry in a host cell, or through blocking viral proteins present on the virus surface as shown in Fig. 1. Subsequently fabricating a targeted nano-system is also beneficial for the elimination of the spreading of viral infection. The detection systems combined with nanoparticles delivered various valuable tools that can be practical for the revealing of pathogenic viral agents for diagnosis purposes. It comprises, though not restricted to, the handling and valuation of nanoparticles, reducing systems dimensions and platforms to utilize nanoscale properties accessible from interfaces between viruses and surfaces.

The current review focuses on the progressive as well as continuing strategies established on magnetic nanoparticles for the specific and non-specific diagnosis including extraction, purification, enrichment, and detection of viruses for combatting fatal infections. Non-specific methods target all types of DNA or RNA regardless of their sequences, while the specific method can target specific sequences of DNA or RNA. The specific method is more appropriate for diagnostic systems as an assay for a specific pathogen. Specific nucleic acid diagnosis is significant where the isolation of a specific sequence of DNA or RNA is preferred. We first

discuss

Limitations of conventional systems

Different analytical assays for separation, extraction, recognition, and observing of virus infections have been developed and are extensively used in repetitive and routine diagnostics applications. Specifically, the separation and recognition of afresh evolving contagious diseases is challenging and thought-provoking. In view of unmet requirements and population expansion, the improvement of nontoxic and active antiviral drugs is a challenging task. Additionally, viruses that are transmitted through travel like dengue viruses, HIV (human immunodeficiency virus), chikungunya viruses, influenza viruses, and HPV (human papillomavirus), or others require diagnostic in addition to therapeutic procedures that inhibit their growth and spread.

Several aspects are required to stop the epidemic spread of a newfangled virus. A well-founded investigation system and detailed information of the noticed variations assist in revealing the danger of an imminent disease and permitting suitable actions to be taken in a period, such as quarantine and invention of a vaccine.

In day-to-day practice, equally molecular biological techniques and quick antigen tests are used, both having their usual pros and cons. In numerous examinations, RT-PCR (real-time polymerase chain reaction) has been established for the most profound and sensitive process in influenza diagnostics. Nevertheless, the better practical exertion, the greater expenses, and a lengthier hands-on time can be disadvantageous in comparison to the rapid antigen test.

Approximations of the sensitivity of diagnostic rapid tests differ importantly in the range between 20% and 90% [START_REF] Kumar | Update on influenza diagnostics: lessons from the novel H1N1 influenza A pandemic[END_REF]. Yet, the patient's choice significantly affects the perceived positive and negative prognostic standards. Even better-quality antigen tests produce a sensitivity of only 79.9% when matched to the PCR test, which decays to 67.3% with time. The quick test can therefore be utilized as a bedside test that delivers the first outcome in minutes with higher diagnostic sensitivity in comparison to PCR [START_REF] Verheyen | Challenges in the diagnosis and prevention of viral infections[END_REF]. Innovation with better-quality potential in the analytical system for viruses is desirable for medical healthcare from diagnostics to therapeutics.

Traditional methods including serologic test, PCR (polymerase chain reaction), and ELISA (enzyme-linked immunosorbent assays) have not rather been complex, however have been lengthy, inefficient, depending on resources, and have restrictions. In Table 2, few methods for amplification of nucleic acid are presently accessible aimed to diagnose contaminations inside laboratory, caused by viruses globally, along with benefits as well as restrictions of them are précised. Therefore, requirement of an accurate and specific system for detection is tremendously growing. To attain this objective, distinctive configurations and approaches have been made to develop a responsive and reliable virus sensor using nanoparticles.

Table 2

Benefits along with shortcomings regarding various traditional systems employed for diagnosis. [START_REF] Sidiq | Benefits and limitations of serological assays in COVID-19 infection[END_REF][START_REF] Ismail | Serological tests for COVID-19 antibodies: Limitations must be recognized[END_REF] RT-PCR has been accepted as an exclusive as well as a reliable test for analyzing and measuring dissimilar RNA within laboratories and medical diagnosis because of eminent precision in the amplification of RNA. Following the existence of coronavirus, many procedures aided RT-PCR to recognize this deadly virus [START_REF] Algaissi | Quantification of the Middle East Respiratory Syndrome-Coronavirus RNA in Tissues by Quantitative Real-Time RT-PCR[END_REF][START_REF] Liu | Positive rate of RT-PCR detection of SARS-CoV-2 infection in 4880 cases from one hospital in Wuhan, China, from Jan to Feb 2020[END_REF]. RT-PCR aided practices have been broadly practiced in COVID-19 detection because of their suitability and accuracy in viral detection and epidemic control but are also persistently delayed because of long procedures as well as labor constraints. Refined extraction and estimation of nucleic acids through complex samples are the foremost necessity to analyze in RT-PCR [START_REF] Tharayil | A short review on nanotechnology interventions against COVID-19[END_REF]. Lower-grade efficacy regarding removal might affect unsuccessful signs via amplification and therefore leads to the wrong diagnosis. Orthodox practices for binding of nucleic acids involve several long steps, also subjected towards impurities [START_REF] Knepp | Comparison of automated and manual nucleic acid extraction methods for detection of enterovirus RNA[END_REF]. To manage and regulate the spontaneous coronavirus increase, conservative methods consumed a lot of manpower as well as greater cross-infections threat. For that reason, fast, convenient, and self-propelled techniques for nucleic acid extraction are enormously essential for detecting coronavirus, but also additional diseases as well.

Method of Detection

Marketable kits and methods, although expensive, are simple to use and typically yield better RNA quality. The accessibility of commercially registered chemical components is also extremely affected due to the interruption of the worldwide supply network as a result of the viral epidemic. Extraordinary price in addition to short accessibility regarding these components execute restricted way towards testing capabilities in both wealthy and unfortunate countries and have contrary side effects associated with extra health problems [START_REF] Holshue | nCo, First Case of 2019 Novel Coronavirus in the United States[END_REF]. Thus, there is pronounced motivation to advance substitute systems or devices that only need nearby, available, and economical chemicals, but also are easy to execute, and competing the depiction of commercially available kits. Above and beyond easing supply deficiency, the unusual means should preferably eradicate the hazard of controlling live viruses, so dropping the severe biological safety necessities upon analysis suitability [START_REF] Wang | Detection of SARS-CoV-2 in Different Types of Clinical Specimens[END_REF]. Any technique of extraction depending on self-building of RNA satisfying the above-stated standards may escalate abilities for testing in scientific practices, nonurban healthcare services, academia labs, as well as field analysis locations [START_REF] Medema | Presence of SARS-Coronavirus-2 RNA in Sewage and Correlation with Reported COVID-19 Prevalence in the Early Stage of the Epidemic in The Netherlands[END_REF]. To address these shortcomings, nanoparticle aided extraction and detection methods are expedient, uncomplicated, and friendly with self-propelled practices [START_REF] Lee | Preparation of iron oxide silica particles for Zika viral RNA extraction[END_REF].

Non-magnetic nanoparticles in viral diagnosis

Nanotechnology can be generally defined as the strategy and usage of numerous nanomaterials and devices for various applications including analysis, handling, regulating, and inhibition of diseases [START_REF] Choi | Nanomedicines: current status and future perspectives in aspect of drug delivery and pharmacokinetics[END_REF][START_REF] Soares | Nanomedicine: Principles, Properties, and Regulatory Issues[END_REF]. Many nanomaterials shown in Fig. 2, have been utilized for extraction and detection of viruses comprising MNPs (magnetic nanoparticles), QDs (quantum dots), SiNWs (silicon nanowires), VLPs (virus-like particles), graphene, inorganic nanoparticles, liposomes, nanoparticles of self-assembled protein, polymers, and metal, silica nanospheres, CNTs (carbon nanotubes), nanostructured surfaces and films [START_REF] Al-Halifa | Nanoparticle-Based Vaccines Against Respiratory Viruses[END_REF][START_REF] Campos | Recent Developments in Nanotechnology for Detection and Control of Aedes aegypti-Borne Diseases[END_REF][START_REF] Zhao | Colorimetric biosensors for point-of-care virus detections[END_REF]. Fig. 2. Schematic view of some nano-magnetic nanoparticles employed to detect various viruses in biological samples (reproduced and modified figure according to Ref [START_REF] Al-Halifa | Nanoparticle-Based Vaccines Against Respiratory Viruses[END_REF][START_REF] Campos | Recent Developments in Nanotechnology for Detection and Control of Aedes aegypti-Borne Diseases[END_REF][START_REF] Zhao | Colorimetric biosensors for point-of-care virus detections[END_REF]).

The employment of nanoparticles aims to take benefits of nanocarriers, eluding their limits. Their appealing properties, such as bioavailability, optimum tunable size, charge, shape, biodegradability, high SRP (surface plasmon resonance), photon exchange, superparamagnetism, luminescence, biocompatibility, immunocompatibility, and tolerability offers an unusual methodology for viral detection. Also, they can easily be ornamented/attached/bonded to different linkers, functional groups, biomolecules, as well as several additional nanomaterials, having the ability to diagnose and treat simultaneously [START_REF] Szunerits | Nanostructures for the Inhibition of Viral Infections[END_REF]. Nano-based detection along with extraction has benefits over outdated methods to permit easy, speedy, highly sensitive, and labelfree detection of viruses leading to a positive influence on public health. This innovative methodology will advance the improvement of regulatory systemization in addition to POC (point-of-care) nanodiagnostic in clinical practice. The nanoparticles usage in such technologies is interesting to recognize, neutralize, and interrupt these pathogens afore they enter into the body. These nanomaterials could be synthesized or acquired from biological sources [START_REF] Singh | The role of nanotechnology in the treatment of viral infections[END_REF].

Continuous efforts have been made for refining the physicochemical characteristics of nanoparticles by surface modifications. Synthetic nano-based formulations played a remarkable role in the diagnosis as a lot of detection systems, based on nanoparticles, show improved wellbeing and efficiency over conventional methods. These nanoparticles have assemblies that resemble pathogens of nanoscale size and stimulate the progression of the human immunity system. Developments of novel nanosystems based on the similarity between synthetic nanoparticles and viruses provide a way out towards persistent and evolving human infections [START_REF] Chattopadhyay | Nanoparticle Vaccines Adopting Viruslike Features for Enhanced Immune Potentiation[END_REF]. Presently, miscellaneous nanoparticles are being exploited for virus detection because of their exceptional properties by different tools, summarized in Table 3.

Table 3

Different non-magnetic nanoparticles and their properties used in the detection of viruses. Nanoparticles propose various prospects in the field of molecular diagnosis. Yet, despite these involvements, it is extremely thought-provoking to securely transform nanoparticles from laboratory invention to clinic. Nanoparticles are very useful for biomedical applications; however, have some bottlenecks which should be addressed to assist their extensive employment in the comprehensive healthcare system. Despite many advantages, they have a contrary side which is the agglomeration or sedimentation of nanoparticles, which optimizes their usage against in vitro viral diagnosis. This problem can be overcome only through proper surface functionalization. Nanoparticles are being employed because of their exceptional as well as novel properties, but researchers across the world have comprehended the importance of stable nanoparticles to develop consistent in vitro biocompatibility models [START_REF] Rai | Nanotechnology-based promising strategies for the management of COVID-19: current development and constraints[END_REF]. An additional concern is the absence of reliable procedures aimed at the biological and physicochemical characterization of nanoparticles [START_REF] Tharayil | A short review on nanotechnology interventions against COVID-19[END_REF]. It is supposed that the physiochemical characteristics of nanoparticles for instance composition, size, shape, charge, crystallinity, solubility, surface area, in addition to surface derivatives for functionalization are risk factors to cause potential toxicity.

Nanoparticles are not themselves a threat. There are only a few aspects that make them risky, specifically their mobility and enhanced reactivity. The ability to extensively produce nanoparticles is another difficulty that is to be overcome for the wider commercialization of nano-based formulations [START_REF] Tharayil | A short review on nanotechnology interventions against COVID-19[END_REF]. Consequently, we should give other perceptions to enhance and upgrade processes and advance more in the transformation of bench-top research to clinical practice.

Another challenge is to understand the genomic and proteomic structure of viruses for their real-time diagnosis. A substantial rate of mutation and consequential genetic multiplicity is also a foremost problem for diagnosis. Viruses do not have adequate targets, hence investigations about the weak points of the virus and susceptibilities of infected cells will allow designing particular ligands that can be used to functionalize nanoparticles to target the virus [START_REF] Abd Ellah | Nanomedicine as a promising approach for diagnosis, treatment and prophylaxis against COVID-19[END_REF].

Moreover, each virus reacts differently from one host to another. Hence, extending the spectrum for nanoparticle-based viral diagnosis is still very challenging. Prominently, with this high rate of virus diffusion, there is a critical requirement to develop a safe and quick method to extract and detect viruses at a reasonable cost. Another challenge is the safety of the workplace using nanoparticles. Health hazards of functionalized nanoparticles should be properly estimated, comprising their fabrication, storing, delivery, applications, possible abuse, and discard.

Applications of nanoparticles enhance the sensitivity, speed, and reliability of, as well as provide more effective options for viral diagnosis; therefore, there is an opportunity that, with more research, it will revolutionize the fight against ongoing COVID-19. Still, numerous aspects need to be considered and a thorough study concerning nanotoxicity should be made for safety and reliability; the requirement for vigorous scale-up synthesis; the biological reactions for nanoparticles containing exposure levels, systemic accumulation, tissue and organ distributions of test living subjects; the possible toxicity of the nanoparticles in the short and long term [START_REF] Zhang | Grand Challenges for Nanoscience and Nanotechnology in Energy and Health[END_REF].

Contribution of magnetic nanoparticles (MNPs) in the diagnosis of viruses

Innumerable inorganic nanoparticles, such as gold, silver, iron oxide, etc., have found their usage in nucleic acid extraction, purification, and detection. Among these, magnetic nanoparticles are extensively consumed for such applications attributed to the high ratio of surface area to volume, excellent dispersion, and extraordinary rate of their bonding to recognition constituents. Their ability to control accumulation magnetically, superparamagnetic behavior, inexpensive preparation, quick isolation inside buffer solutions, along with detection of the signal makes purification, pre-concentration, and separation regarding nucleic acids quick and specific [START_REF] Kang | Surface design of magnetic nanoparticles for stimuli-responsive cancer imaging and therapy[END_REF][START_REF] Jamshaid | Magnetic particles: From preparation to lab-on-a-chip, biosensors, microsystems and microfluidics applications[END_REF]. MNPs are being used by researchers to immobilize proteins, antibodies, enzymes, and more bioactive substances for speedy and effectual biomolecules separation utilized for targeting [START_REF] Hedayatnasab | Review on magnetic nanoparticles for magnetic nanofluid hyperthermia application[END_REF]. In complex samples, biomarkers can be enriched and preconcentrated, Functionalization and surface coating by oxiranes, carboxylic acids, amines, in addition to aldehydes are generally employed for immobilization of enzymes, proteins, nucleic acids (RNA and DNA), plus bioanalytes onto the surface of nanoparticles covalently [START_REF] Riemann | Comparison of manual and automated nucleic acid extraction from whole-blood samples[END_REF]. They possess both magnetic particles and nanoparticles characteristics so can easily be manipulated by external magnetic fields. MNPs reduce the time of relaxation of protons regarding water molecules, as a result, contrast is generated that has also been utilized to detect viruses [START_REF] Chen | One-step detection of pathogens and viruses: combining magnetic relaxation switching and magnetic separation[END_REF]. On account of biocompatibility, exceptional dispersion properties as well as suitable chemical functionality aimed at fixation, it is possible to employ magnetic nanoparticles bonded to different elements for recognition to captivate biomolecules, to isolate support magnetically from the mixture of reaction, and also as compact adsorbent [START_REF] Zhuang | Polymerase chain reaction combined with fluorescent lateral flow immunoassay based on magnetic purification for rapid detection of canine parvovirus 2[END_REF].

MNPs accumulation and dispersability could easily be regulated by applying a magnetic field externally. Nonmagnetic particles bonded to MNPs remain suspended in solution uniformly when there is no field. On application of magnetic field; these particles get isolated. Active molecules for instance bio-adsorbents and functional ligands bonded with MNPs possibly are attached with particular biomolecules, for instance, proteins, DNA, and enzymes, and could be partitioned via a magnetic field which is applied externally. The aforementioned technique offers exceptional specificity, fast isolation as well as significant reproducibility that paved the way for a scientific revolution in biological research [START_REF] Mauk | Simple Approaches to Minimally-Instrumented, Microfluidic-Based Point-of-Care Nucleic Acid Amplification Tests[END_REF]. Finite-size, surface coating, and magnetic

properties have a substantial impact on viral diagnosis. Nanoparticle size shows the possibility of nanoparticle interaction with the outer environment [START_REF] Williams | The application of magnetic nanoparticles in the treatment and monitoring of cancer and infectious diseases[END_REF]. As the size of MNPs decreases, there is a greater probability that atoms confined inside the nanoparticle will become surface atoms and with a higher percentage of surface atoms. Magnetic properties of MNPs also decrease with that reduces their efficiency for detection [START_REF] Issa | Magnetic nanoparticles: surface effects and properties related to biomedicine applications[END_REF]. The instability of magnetic nanomaterials makes it important to develop approaches to stabilize MNPs against degradation and enhance biocompatibility. Such strategies frequently include modification of the surface with an organic layer or inorganic layer [START_REF] Kush | Aspects of high-performance and bio-acceptable magnetic nanoparticles for biomedical application[END_REF]. The surface functionalization of MNPs governs the binding efficacy of nucleic acids. Different materials or polymers have been used to modify the surface of MNPs for instance silica, dextran, polystyrene, or poly (vinyl alcohol) (PVA), polyethylene glycol (PEG) to maintain their stability and enhance their performance for diagnosis [START_REF] Jat | Magnetic nanoparticles: an emerging nano-based tool to fight against viral infections[END_REF]. The coating of MNPs reduces the risk of opsonization and also their neutral pH excludes the functional group's conjugation. Hence, synthetic polymers, for instance, PAA (poly (acrylic acid)) are favored for their capability to bind to biological macromolecules, such as nucleic acids [START_REF] Kush | Aspects of high-performance and bio-acceptable magnetic nanoparticles for biomedical application[END_REF] Suitable surface coating of MNPs is necessary, which not only gives colloidal stability to the nanoparticles but also maximizes the possibilities for use in various other applications. The colloidal stability of MNPs under physiological conditions, for instance, at pH~7.4, in blood, in physiological salts, and protein concentration is the minimum requirement for biomedical applications [START_REF] Socoliuc | Magnetic Nanoparticle Systems for Nanomedicine-A Materials Science Perspective[END_REF]. Therefore, the agglomeration of MNPs has to be stopped via a surface coating, either during or after synthesis. The molecules or functional groups bonded covalently can more improve colloidal stability than adsorbed ones. An optimized coating on MNPs surface is required to interact with nucleic acids. The coating layer of core-shell nanosystems provides optimum hydrophobicity/hydrophilicity in a given medium and active sites for anchoring bio-functions [START_REF] Socoliuc | Magnetic Nanoparticle Systems for Nanomedicine-A Materials Science Perspective[END_REF]. Some properties of magnetic nanoparticles including their size and composition used in viral diagnosis have been summarized in Table 4.

Extraction and purification of nucleic acid

One elementary constituent of life is "nucleic acid" which is tremendously vital for physiological processes taking place inside the body. They primarily transfer, in addition to retaining, genomic information that assisted in detecting mutation in genes linked to some specific health situations. Its isolation and decontamination are significant in molecular biology.

Isolation of RNA and DNA is a phase before diverse biochemical and diagnostic processes like replicating, recognition, intensification, hybridization, sequencing, and DNA production [START_REF] Stray | Isolation of Cell-Free DNA from Maternal Plasma[END_REF].

The extraction of nucleic acid by traditional methods was lengthy and laborious. It contains a multiplex procedure of removal and centrifugation that is severely controlled by low clarity and small yields of the separating samples [START_REF] Stray | Isolation of Cell-Free DNA from Maternal Plasma[END_REF]. Early nucleic acid extraction and purification are significant for inhibiting, treating, as well as recognizing the disease. In the previous era, MNPs have been used to extract nucleic acids that are present in the lysed samples by their adsorption on MNPs because of the existence of modified functional moieties on MNPs surface [START_REF] Kharat | Multifunctional Magnetic Nano-platforms for Advanced Biomedical applications: A Brief Review[END_REF]. Extraction and purification based on MNPs are free of centrifugation and have been recognized as easy, simple, controllable plus attuned with mechanization as well as scaled-up procedure [START_REF] Pichl | Magnetic bead technology in viral RNA and DNA extraction from plasma minipools[END_REF][START_REF] Varadi | Rapid magnetic bead based sample preparation for automated and high throughput N-glycan analysis of therapeutic antibodies[END_REF].

The modified MNPs adsorb and separate the nucleic acid quickly from contaminated lysis solution employing an externally applied magnetic field. Afterward, nucleic acids are further separated from the functionalized MNPs by the desorption route in the eluent utilizing this methodology. This system is although much smoother and faster than orthodox techniques, still, it is multi-step, which is inadequate for applied detection [START_REF] Ali | Development of Magnetic Nanoparticles Based Nucleic Acid Extraction Method and Application in Hepatitis C Virus Chemiluminescent Detection[END_REF].

Techniques based on MNPs have established significant concern due to their expedient manipulation, low cost, and easiness of automation. Various aspects comprising lysis and washing solutions (buffers, MNPs, ethanol) would affect nucleic acid production. The mean RNA and DNA yield attained through 1 mL of Hep G2 having approximately 10 6 cells have ranged in between 9.7-14.7 μg at A260/A280. Simultaneous extraction of RNA or DNA from cancer cells consuming silica-coated MNPs was appropriate for future acts for instance, such as PCR as well as RT-PCR [START_REF] Wang | Simultaneous Extraction of DNA and RNA from Hepatocellular Carcinoma (Hep G2) Based on Silica-Coated Magnetic Nanoparticles[END_REF]. Immobilization of nucleic acid via SPRI (Solid-phase reversible immobilization) onto MNPs proposes an elegant alternate for approaches based on centrifugation or else columns [START_REF] Haddad | DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering[END_REF]. During drying, it is feasible to reversibly bind MNPs inside the sample with nucleic acids. These nucleic acids are promptly isolated from contaminations by the magnetic field, and as a result, purified nucleic acids with a dissimilar ionic strength are freed from MNPs via eluting buffer after several fast washing steps as shown in Fig. 4 [START_REF] Oberacker | Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation[END_REF]. SPRI permits exhaustive washing steps for eradicating inhibitors in reactions taking place in molecular biology in addition to producing next-generation sequencing and better-quality RNA for PCR.

Extraction of RNA based on MNPs has been integrally accessible, extensible, as well as responsive towards mechanization following no centrifugation besides the usage of low-cost materials. Though considerably quicker besides easier when compared to procedures based on spin column, still further, such approaches to extract comprise of manifold treating phases including lysis, bonding, rinsing, plus elution, that enhances operative complications in real-time clinical diagnosis. opposes kits that are commercially available for detecting influenza, SARS-CoV-2, as well as RSV (respiratory syncytial virus). As this approach is centrifugation-free, therefore continuing and future research will be majored in self-regulating high-throughput of RNA extraction by liquid-handling robots. To put it concisely, MAVRICS has a great potential to develop a qualifying methodology for prevalent community analysis and wastewater management in the present-day and future epidemics [START_REF] Ramos-Mandujano | A Robust, Safe, and Scalable Magnetic Nanoparticle Workflow for RNA Extraction of Pathogens from Clinical and Wastewater Samples[END_REF]. An automatic rapid nucleic acid extractor was developed using MNPs for processing 16 samples instantaneously. This system is simple and friendly to operate and completes the process of nucleic acid extraction in 30 minutes. The yield and purification of 16 samples from one hepatic cellular cancer (HCC) sample propose that the developed system is stable and reliable [START_REF] Chen | Design and Application of Automatic and Rapid Nucleic Acid Extractor Using Magnetic Nanoparticles[END_REF].

Nucleic acids separation by magnetic nanoparticles revealed several benefits over the challenging methods, consenting to segregate nucleic acids directly from non-prepared samples, with culture media, water, physiological liquids, as well as blood, etc. Practically, sample volumes offer no limitations on this technique and can be taken comparatively selectively and effortlessly even from viscous complex utilizing the capability to alter the magnetic characteristics of nanoparticles [START_REF] Berensmeier | Magnetic particles for the separation and purification of nucleic acids[END_REF].

Enrichment/concentration of target substance

Viruses pose a continuous danger for health globally. Therefore, enriching target analytes rapidly has been critical and essential that are present in low concentrations in complex matrixes to detect the viruses. A methodology was proposed that integrates MRS (magnetic relaxation switching) with MS (magnetic separation) to detect pathogens in single step along with extreme reproducibility and sensitiveness. In this method, magnetic field with strength 0.01 Tesla was applied that part out large MNPs with diameter of 250 nm from small MNPs having diameter of 30 nm. T2 (transverse relaxation time) of molecules of water surrounded by small MNPs was used to read signals in such assays as shown in Fig. 5. The proposed approach assimilates enrichment, extraction, also detection of target analyte in a single step. The whole process of immunoassay was finished within thirty minutes. It showed improved reproducibility, good sensitiveness, as well as appropriate procedure, consequently offers platform towards POC investigation in comparison to tradition sensors [START_REF] Chen | One-step detection of pathogens and viruses: combining magnetic relaxation switching and magnetic separation[END_REF]. HAV (Hepatitis A virus) remains primary source that results in hepatitis. Approaches for recognizing HAV are very inadequate. To attain this objective, protamine (P) was bonded to MNPs surface possessing diameter of 20 to 30 nm covalently followed by chemical reaction that was carried out in three steps. When utilized to concentrate HAV out of milk (40 ml), PMNPs (50 μl) were introduced in sample. After 20 minutes gentle rotation, there was a capturing of PMNPs by a magnet continued for thirty minutes. PMNPs that were seized were rinsed by buffer solution of glycine (NaCl-0.14 M, glycine-0.05 M, Tween 20-0.2% (v/v)) at pH of 9.

Extraction of HAV-RNA was performed utilizing "QIAamp MinElute Virus Spin Kit" followed by its measurement via simultaneous RT-PCR. This technique presented LOD (limit of detection) equals to 8.3 × 100 PFU (Plaque forming units) regarding HAV present inside milk. This entire process designed for concentration possibly was accomplished within fifty minutes.

This established system has been affordable as well as simple to recognize [START_REF] Wu | Concentration of hepatitis A virus in milk using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles[END_REF]. Carboxylated MNPs have also been used for immobilization of target; HBsAg (hepatitis B surface antigen) aimed at quick separation magnetically. The selected aptamers were used to develop a chemiluminescence aptasensor based on magnetic separation and immunoassay to detect HBsAg from pure protein or actual serum samples with LOD of 0.1 ng/mL, considerably less than LOD measured by an ELISA (0.5 ng/mL). The concentration of HBsAg is directly proportional to chemiluminescent intensity with range starting from 1 ng/mL to 200ng/mL [START_REF] Xi | Selection of HBsAg-Specific DNA Aptamers Based on Carboxylated Magnetic Nanoparticles and Their Application in the Rapid and Simple Detection of Hepatitis B Virus Infection[END_REF]. SDA (strand displacement amplification) in combination with magnetic nanoparticles and electrochemiluminescent (ECL) nanospheres (EN), "enrichment-stowage-cycle" approach has been suggested for developing biosensor based on electrochemiluminescence to recognize DNA of HIV. H1-MNP (H1-Capture hairpin DNA) conjugates efficiently enrich as well as isolates DNA out of composite matrix straightly, shortening operational period along with procedure. This process enhanced ECL signals close by 11.3 fold. The signals were more amplified by SDA nearly 3.77-fold. MNPs incorporation boosted specificness for detection as well as constancy, driving the above-mentioned scheme suitable for real-world utilizations. Efficacious recognition regarding HIV-DNA inside composite samples, for instance in milk, FBS (fetal bovine serum), urine, plus blood) showed its probability for diagnosis on time as well as precise quantification of biomarkers present in actual samples with low concentration [START_REF] Wu | Enrichment-Stowage-Cycle Strategy for Ultrasensitive Electrochemiluminescent Detection of HIV-DNA with Wide Dynamic Range[END_REF]. Enriching concerning target compounds, impurities exclusion, in addition to decontamination of recognized analytes offers pronounced accuracy along with opportuneness intended for future research.

Detection of nucleic acid

Effectual RT-PCR analysis necessitates quantifiable nucleic acid removal out of different specimens that maintain extraordinary pureness. Lower efficiency for extraction and quality possibly will have variation in inhibitors of PCR that generates poor signals, resulting in incorrect negative outcomes due to unreliable readouts in the time of amplification. Accordingly, speedy, suitable, and mechanized nucleic acid extraction systems are extremely required for observing and avoidance of infectious diseases [START_REF] Zhao | A simple magnetic nanoparticles-based viral RNA extraction method for efficient detection of SARS-CoV-2[END_REF].

A fast and precise detection technique was developed to investigate RSV and ADV types of respiratory viruses coming out of identical testing analytes. It was comprised of twofold steps: co-extraction of DNA along with RNA taken out of mucus, followed by complicated RT-PCR.

The instantaneous nucleic acid co-extraction process established on MNPs and the viral DNA

and RNA amplification from mucus technologically advanced a complex RT-qPCR mode. This MNPs-based technique presented improved co-extraction effectiveness regarding DNA as well as RNA, in addition to an evaluation in distinct clinical samples. The time requisite was shortened, and the approach simplified preparation of a sample aimed at PCR analysis i.e. entire preparation of sample besides separation of nucleic acid possibly completed within thirty minutes. MNPs utilization in addition to prevention concerning centrifugation formed the basis of computerized co-extraction of nucleic acids including RNA as well as DNA. An easy method along with the prevention of using organic solvents and also having displeasing smells mark it appealing to be used for repetitive analytical labs, particularly to detect pathogens responsible for respiratory diseases through complex RT-qPCR. Whereas it indicates initial exploration, more research on clinical specimens is requisite eventually [START_REF] He | Integrated DNA and RNA extraction using magnetic beads from viral pathogens causing acute respiratory infections[END_REF].

Silica-coated MNPs are broadly used to extract out of different biological specimens. A vigorous and innovative Fe 3 O 4 @Silica (silica-coated magnetite particles) was employed to extract RNA from samples of serum obtained from the ZIKA virus. After the synthesis of MNPs, these nanoparticles are functionalized by silica through TEOS (tetraethoxysilane) hydrolysis in a basic medium. By purely dispersing agglomerates of magnetic particles in a solution of GdnHCl (guanidinium chloride), the surface of particles coated with silica was able to bind RNA. Efficacy of extracting RNA had been assessed through removing RNA out of serum taken from ZIKA virus, further monitored via PCR. In addition to the elimination of inhibitors of PCR, statistics specify outstanding recapture of target nucleic acid (RNA). A developed system of silica functionalized MNPs offers economic, operative, and equipped for a scale-up method whose execution is comparable to profitable substitutes to prepare samples regarding DNA along with RNA. The budget required for medical examinations is possibly reduced by hundred times employing practically accessible MNPs conjugated with prepared material aimed at extraction of the virus [START_REF] Lee | Preparation of iron oxide silica particles for Zika viral RNA extraction[END_REF].

A different system for immediate detection of many viruses was suggested. This scheme has verified high efficacy and appropriateness for handling clinical test specimens. In addition, a magnetic isolation method was accustomed through the trial, which started through the extraction of a nucleic acid towards chemiluminescence recognition. On account of the mentioned positive and active adoption of the MNPs, this evaluation can be adapted into an automatic setup for the real-time revealing of numerous infections that will additionally decrease the cost of detection per pathogen. The manipulation of MNPs functionalized with silica for extracting nucleic acid and MNPs functionalized with carboxylic groups in chemiluminescence transformed the scheme to an automatic system to scale-up high throughput strategies. The above-mentioned analysis also provided an alternate way to examine clinical samples in blood screening components [START_REF] Ali | Simultaneous detection of multiple viruses based on chemiluminescence and magnetic separation[END_REF]. Single-step complex RT-PCR had been conducted for intensifying nucleic acids extracted from the virus. Diverse probes stabilized by amino groups to capture target sequences regarding HCV, HBV, as well as, HIV had been distinctly bound to MNPs surface functionalized by carboxylic groups as shown in Fig. 7. Incubation of these probes functionalized MNPs with RT-PCR products labeled with biotin inside alternating tubes assist in detaining particular sequences of HCV, HBV, else HIV. SA-AP (Streptavidin-modified alkaline phosphatase) gets attached to biotin. Lastly, CL (chemiluminescence) had been measured after addition of AMPPD (3-(2′-spiroadamantane)-4-methoxy-4-(3′-phosphoryloxy)phenyl-1,2dioxetane) [START_REF] Ali | Simultaneous detection of multiple viruses based on chemiluminescence and magnetic separation[END_REF]. Viral-induced nanoassembly of MNPs was fabricated from monodisperse MNPs bonded to virus-surface-specific antibodies in the existence of particular particles of viruses. They exhibited improved magnetic properties and were distinguished via magnetic resonance methods (NMR/MRI) which exactly permitted highly selective and sensitive viral detection within the composite biological medium for instance suspension of cells, blood, lipid emulsions, cultural media, as well as complete tissue. The established scheme can precisely identify herpes virus-1 in addition to adenovirus-5, at a concentration of 5 particles of virus present in 10 microliters, and not requiring PCR amplification and massive preparation of samples. It is also self-regulating, not depending on optical characteristics of the solution, permitting viral detection in compound unclear solutions. The labeled analysis represents a delicate process as compared to contemporary ELISA, also the bonding of virus to surface of solid is not required. Moreover, this technique presents a great potential for detection of HSV and ADV viruses circulation by in vivo MRI [START_REF] Perez | Viral-induced selfassembly of magnetic nanoparticles allows the detection of viral particles in biological media[END_REF].

Fluorescent MNPs were utilized to identify avian influenza viruses including H1N, H9N2, as well as H7N9 instantaneously. Superparamagnetic nature along with intense magnetic nanoparticle reaction could efficiently trap targets and then separate them requiring no pretreatment of the sample. These MNPs will be used for counting distinct particles aimed at complex detection accurately. The aforementioned process possesses easy steps for amplification of the signal, unusual sensitivity for detection, as well as pronounced perspective to diagnose different diseases at early stages [START_REF] Wu | Digital Single Virus Immunoassay for Ultrasensitive Multiplex Avian Influenza Virus Detection Based on Fluorescent Magnetic Multifunctional Nanospheres[END_REF].

RT-LAMP (Reverse-transcription-Loop-mediated isothermal amplification) has also been combined with carboxyl modified magnetic nanoparticles for detection of H7N9 RNA of influenza virus. It includes the procedure of CL, where molecules used as probes get bounds to ALP (alkaline phosphate) enzyme modified with streptavidin. Chemiluminescence signal was recorded indicating virus presence inside the sample. The method of extracting viruses gets rationalized by employing this methodology in RT-PCR. It permits better recognition consuming not as much of time. Additionally, it grasps significant potential to detect and analyze viruses, specifically avian influenza virus [START_REF] Wang | Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence[END_REF]. An innovative system for POC testing formulated on MNPs was developed for automatic real-time adenovirus detection. The results obtained by this experiment and from commercialized systems were similar, verifying system reliability [START_REF] Chen | Performance Evaluation of a Novel Sample In-Answer Out (SIAO) System Based on Magnetic Nanoparticles[END_REF]. An economical immunoconjugate magnetic nanosystem was developed to detect NS1 protein of dengue virus calorimetrically, consuming NS1 antibodies that were attached to MNPs (magnetite) surface covalently. The developed system has ability to identify NS1 in case of infection or reinfection. The given process possibly modified to detect proteins related to evolving infections caused by viruses, in particular existing COVID-19 epidemic [START_REF] Ramírez-Navarro | A magnetic immunoconjugate nanoplatform for easy colorimetric detection of the NS1 protein of dengue virus in infected serum[END_REF].

Magnetic nanoparticles in diagnosis of coronavirus (COVID-19)

Molecular diagnostics and investigation of pathogenic viruses for instance coronavirus are based on extraction and segregation of nucleic acids. Epidemics at large scale caused worldwide deficiency concerning registered marketable chemicals as well as bio-safe labs for securely achieve analysis. Hence, early detection of nucleic acids is immediately desirable to address the problems.

Currently, detection of COVID-19 based on RT-PCR is comprehensively used. This is a well-timed and accurate diagnostics procedure but is still constrained due to the long duration and extensive manpower necessity. To deal with this difficulty, the modification of MNPs and procedure to extract RNA aimed at probable recognition of coronavirus was reported.

Nanoparticles of zinc ferrite had been synthesized using combustion, followed by functionalization with poly (vinyl alcohol) modified with silica and carboxylate. Characterization of prepared MNPs verified properties as well as functionalities at nano-scale. The suggested methodology was able to isolate and extract the viral RNA from some samplings by a computerized system. Taking comfort and skillfulness into consideration, this procedure may pointedly reduce the time of operation regarding existing diagnosis of coronavirus at molecular level [START_REF] Somvanshi | Multifunctional nano-magnetic particles assisted viral RNA-extraction protocol for potential detection of COVID-19[END_REF]. Zinc ferrite (ZNF) magnetic nanoparticles had also been synthesized via economical sol-gel process that was auto-combustive. They were further functionalized by polymers having with carboxylic groups (CPoly) as shown in Fig. 8. Among magnetic nanomaterials, zinc ferrite (ZnF) was selected owing to its extraordinary chemical stability, soft magnetic behavior, easy preparatory methods, and biocompatible nature [START_REF] Kmita | Use of Specific Properties of Zinc Ferrite in Innovative Technologies[END_REF]. In view of influential interactions of carboxylic groups with nucleic acids, modified MNPs enable prompt in addition to prospective RNA adsorption. It is a facile and provident procedure and an efficient alternate for prevalent methodologies [START_REF] Somvanshi | Multifunctional nano-magnetic particles assisted viral RNA-extraction protocol for potential detection of COVID-19[END_REF]. The multifunctional chitosan-coated lithium zinc ferrite MNPs integrated with graphene oxide magnetic nanocomposites (CHLZFO-GO MNC) were well produced by sol-gel auto combustion and ultrasonication. In this method, zinc-doped lithium ferrites were first coated with biopolymer (chitosan) and then incorporated into sheets of GO to introduce carboxyl groups. The prepared nanocomposites were spherical in shape and crystallite sizes were found in the nanometric range (~ 19 nm to 35 nm). The experimental magnetization values are appropriate for the effectual and vigorous extraction procedure of RNA leading to potential detection of SARS-CoV-2 in COVID-19 patients. The protocol for extraction includes a combination of lysis/binding buffer with CHLZFO-GO MNCs in a single step, followed by washing, elution, and to end assembling the extracted RNA for RT-PCR. The CHLZFO-GO/RNA complex can be used directly for RT-PCR reaction without the elution step. Hence, the likelihood of getting false-negative results in RT-PCR protocol can be more reduced. The RT-PCR method combined with these MNC is easy, reasonable, time-saving, and many operatives and thus can be seen as a probable substitute for conventional RT-PCR techniques. MNC-based extraction can be an inventive technology to isolate RNA from swabs for fast COVID 19 tests at a larger scale [START_REF] Singh | Fabrication of chitosan-coated mixed spinel ferrite integrated with graphene oxide (GO) for magnetic extraction of viral RNA for potential detection of SARS-CoV-2[END_REF].

The existing epidemic of unusual coronavirus disease pulls wide-reaching fears because of its extensive incubation time and durable contagion. Due to this, there is an undeniable necessity for quick and consistent detection of the pathogens of SARS-CoV-2 to prevent its spread. Magnetic biosensors especially the giant magnetoresistive (GMR) biosensor along with MNPs can be very sensitive and promising biosensing devices for rapid detection of SARS-CoV-2 S-protein and +ssRNA obtained from biological samples (blood, urine, serum, etc.). This is an alternate route to develop for sorting out COVID-19 patients and thus protect the outbreak of COVID-19 to regain a normal pace in the world [START_REF] Islam | Plausible Approach for Rapid Detection of SARS-CoV-2 Virus by Magnetic Nanoparticle Based Biosensors[END_REF]. The potential activity of MNPs on SARS-CoV-2 and HCV by molecular docking studies was also investigated. The proposed model revealed that iron oxide nanoparticles particularly Fe 2 O 3 and Fe 3 O 4 interacted efficiently with SARS-CoV-2 S1-RBD (receptor binding domain) and HCV (Hepatitis C virus) glycoproteins, E1 and E2. It was established that Fe 3 O 4 made a more stable complex with S1-RBD while for HCV E1 and E2, a more stable complex was made with Fe 2 O 3 . Thus these revealed interactions to be related to configurational changes in viral structural proteins and succeeding virus inactivation. This showed the potential application of MNPs to control diverse viral infections [START_REF] Abo-Zeid | A molecular docking study repurposes FDA approved iron oxide nanoparticles to treat and control COVID-19 infection[END_REF].

Even though molecular analysis depending on PCR has been extensively practiced in medical analysis presently, still well-timed and precise diagnosis is restricted on grounds of Poly-amino magnetic nanoparticles (Poly-NH 2 -MNPs) were prepared by functionalizing MNPs by polymer having negative charge to extract and purify RNA of viruses, which possibly be executed for detecting coronavirus substantially. MNPs were synthesized with simple as well as economical co-precipitation technique. This is followed by process of hydrolysis consuming APTES (3-aminopropyl triethoxysilane). The poly (amino ester) was prepared through polymerization of diacrylate-amine employing 1,4-butanediol diacrylate and 6-aminocaproic acid. In last, Michael addition methodology was used to coat amino-MNPs (NH 2 -MNPs) by the poly (amino ester) [START_REF] Payra | On-water magnetic NiFe2O4 nanoparticle-catalyzed Michael additions of active methylene compounds, aromatic/aliphatic amines, alcohols and thiols to conjugated alkenes[END_REF]. Entire coating of MNPs with polymer to yield Poly-NH 2 -MNPs gives preferred negative charge to extract RNA properly, as displayed in Fig. 10. Synthetic procedure allows implementation around 50,000 RT-PCR tests simultaneously to detect coronavirus. However, no reduction in signal obtained by RT-PCR was observed with increase in nanoparticle concentration present in solution. So it is requisite to perform additional investigations by altering or modifying size, type, in addition to coating of MNPs with the aim of getting an optimized material to enhance the response of application in qRT-PCR. But this systematic protocol can be used as a benchmark for extracting RNA of better quality afore their possible execution in PCR. This provided a solution to the recognition along with regulation of eruptive spread regarding novel coronavirus although budget restrictions of the country in addition to availability/facility of laboratory or chemicals throughout this confirmed: "International Public Health Emergency" [START_REF] Chacon-Torres | Optimized and scalable synthesis of magnetic nanoparticles for RNA extraction in response to developing countries' needs in the detection and control of SARS-CoV-2[END_REF]. In another method, the SARS-associated coronavirus gene was detected by consuming functionalized MNPs along with a PCR assay. First functionalized silica-coated SPIONs were used to capture and enrich target complementary DNA (cDNA) of SARS linked coronavirus from the mixture of target cDNA and non-target cDNA. Moreover, the enriched target cDNA were amplified via common symmetry PCR. After that, it was particularly isolated from the double strands PCR products by application of silica coated SPIONs again. Lastly, the amplified target cDNA was detected using silica coated fluorescent nanoparticles as signaling probes. The results indicate that the target cDNA can be examined effectively with LOD of 2.0 x 10(3) copies. Non-specific amplification was also inhibited through this method. Additionally, this procedure is fast and promising for viral detection [START_REF] Gong | Combination of Functionalized Nanoparticles and Polymerase Chain Reaction-Based Method for SARS-CoV Gene Detection[END_REF].

Consistent biosensing based on MNPs is one of the most promising approaches for rapid and highly sensitive detection of biomolecules to control the outbreak of SARS-CoV-2. In another approach, SARS-CoV-2 with was detected using functionalized MNPs through measuring their magnetic response in an AC magnetic field. For this method, mimic SARS-CoV-2 containing spike proteins and polystyrene (PS) beads are utilized for experiments. Functionalized MNPs were employed as sensors to detect a mimic virus consisting of 100 nm PS beads integrated with SARSCoV-2 spike proteins (Fig. 11). Experiments on AC susceptibility (ACS) spectra and magnetic particle spectroscopy (MPS) signal of samples with different mimic virus concentrations were performed. Results showed that the binding performance between mimic SARS-CoV-2 and functionalized MNPs enhances the effective Brownian relaxation time and changing the MPS signal. Suggested approach permits the quick detection of mimic SARS-CoV-2 with LOD of 0.084 nM (5.9 fmole). This method has pronounced potential to design lowcost and POC device for sensitive diagnostics of SARS-CoV-2 [START_REF] Zhong | Toward Rapid and Sensitive Detection of SARS-CoV-2 with Functionalized Magnetic Nanoparticles[END_REF].

Conclusions and perspectives

Currently, nanotechnology focuses to develop strategies and protecting from catching the virus in the first place. The usage of nanoparticles, for extracting nucleic acids, enrichment of target analytes, in addition to detection of nucleic acids, offers the huge possibility to advance process that is presently accessible for diagnosis, or else advancement regarding different nanobased technologies. They both are urgently prerequisites for multiplexed detection and early diagnosis of diseases. The excellent features of nanoparticles, comprising their strong electrochemical and optical properties, biocompatibility, controllable sizes, and costeffectiveness play a vital role in a broad range. These properties can be easily tuned by surface functionalization using various substrates, offering incredible potential for practical applications.

Nanoparticles used for viral diagnosis offer a more sensitive, specific, economic, and easy to use diagnostic kits for detecting the pathogenic virus in comparison to conventional techniques. Thus engineered magnetic nanoparticles unified with conventional antiviral properties through multi-functionalizing are favorable materials designed for research in the biomedical field in addition to clinical practice. Certainly, an excessive effort is a prerequisite for presenting transportable as well as reusable strategies having the ability to categorize viruses along with better precision and accuracy. Ultimately, progress in the field of nanotechnology will turn out to be an evident invention for medication for ceasing the spread of diseases caused by viruses and also offer a healthy and better life to possibly infected patients. The integration of magnetic nanoparticles into a disposable lateral flow test platform is also in focus to screen patients for the COV1D-19 virus. Recently not any lateral flow test particular to coronavirus strain is available. They do not require capable technicians as well as supplementary lab apparatus to be used as screening equipment, assisting grouping of individuals. Researchers are soon to propose more precise, economical, also handy kits for diagnosis to identify viral pathogens including the novel coronavirus relying on exclusive properties of MNPs. Efforts are also in the process to explore the potential of MNPs to obtain better sensitivity, selectivity and simultaneous detection. They considerably participate to detect, diagnose, and understand, in addition to treat diseases because of viruses. It also assists to reduce viral eruptions in future.

MNPs need to be upgraded to meet application necessities rapidly for extraction and detection in magnetic field externally applied. Stability regarding MNPs directly affects its application therefore MNPs should be modified with suitable functionality that increases surface compatibility along with efficiency for bonding ligands. Presently, due to limited available information in regards to basic as well as clinical examination, current options for treating and managing coronavirus includes new nanoparticles and therapeutic drugs. Though, attention is required before forming any premature elucidations because still scientific testings in clinics are under way, plus temporary experimental statistics are not being available yet. Need for an accepted as well as effectual vaccine to treat existing coronavirus, aim is tracking along with diagnosing diseases easily in early phases. This forces medical precaution to move from reactive to proactive form. For early detection of viral infections, an urgent need is to develop new, fast, effective, and applicable technologies in terms of real-time diagnosis. The incorporation of MNPs with molecular diagnostic systems can be used to develop fully-automatic and closed nucleic acid detection assays. With enhanced sensitivity and less required time to detect, there would be prominent impact in biomedicine, detection at clinical level, together with other biomedical areas. Nevertheless, as researchers working with nanoparticles, we consider that only by operative and close by association between various stakeholders of society will we be able to combat current pandemic as well as to react rapidly to any future global health emergencies.

In future prospective, FDA-approved-MNPs should be employed into clinical trials for viral diagnosis. Moreover, due to their biocompatibility, MNPs should be used for manufacturing antimicrobial fabrics for instance coats, gloves, masks, head nets, bed sheets, overshoes, and, pillow covers. This will be an advanced measure to control viral infections in hospitals. There is a need of simple and cost-effective nanomaterial-based products for the prevention, diagnostics and treatment of viruses. Through research and development, nanotechnology could help in refining diagnostics sensitivity using only a small amount of biological sample. The conventional methods could also be improved to activate host immune response against the viruses. One of the main challenges is to guarantee the safe usage of nanomaterials. The employment of biodegradable nanoparticles is important to ensure their usage inside human body. In vivo studies should also be more investigated to understand the toxic behavior of nanoparticles for long term use in human body. An operative methodology for large-scale synthesis with accurate control over size, surface modifications and other parameters at minimum cost is critical particularly for early diagnosis. We visualize that nanotechnology is a powerful tool to combat ongoing COVID-19 and more investigations are essential to add new systematic and methodological knowledge to

  limitations regarding conventional techniques along with the contribution of different nanoparticles for virus detection and later move towards prominence concerning the employment of magnetic nanoparticles (MNPs) for point-of-care (POC) testing for nucleic acid extraction, enrichment, and detection.

Fig. 1 .

 1 Fig. 1. Prospects of nanotechnology for prohibiting the spread of viral infection.

Fig. 3 .

 3 Fig. 3. Morphology, functionalization, surface coating, and properties of MNPs.

  potential regarding MNPs viral diagnosis, we described extraction, purification, concentration, and detection of nucleic acid employing MNPs in the following sections.

Fig. 4 .

 4 Fig. 4. Schematic procedure for nucleic acid purification by means of MNPs.

Fig. 5 .

 5 Fig. 5. Representation of MS-MRS sensor. Magnetic beads of 250 and 30 nm diameter precisely

  Magnetic nanoparticles have also been used for detaching or else separating particles of virus, as an alternative of extraction viral nucleic acid. Enriched particles of virus intensely bounded with MNPs due to the presence of functionalized receptors designed for targeting. Virus particles gained magnetic property after bonding to SPIONs (superparamagnetic iron oxide nanoparticles). Afterwards, virus get detached taking advantage of these SPIONs using magnet coupled with cell-reliant tittering analysis, qRT-PCR (quantitative reverse transcriptionpolymerase chain reaction) test, in addition, immunochromatographic test strips. Biomimicry of SPIONs enriches virus inside samples to improve virus detection as revealed in Fig. 6 [93].

Fig. 6 .

 6 Fig. 6. An illustration representing biomimicry of MNPs that mimic the cell membrane of the

Fig. 7 .

 7 Fig. 7. Probes functionalized with amino groups, to capture target sequences of HCV, HBV, as

Fe 3 O 4 /

 34 SiO 2 (magnetite nanoparticles coated by silica), as well as optimal buffers, have also been used to separate two sorts of genomic DNA including Epstein-Barr virus (EBV) together with hepatitis virus type B (HBV), to further recognize viruses present in serum by PCR. Acquired preliminary statistics specify that Fe 3 O 4 /SiO 2 nanoparticles delivered improved sensitivity to detect them, in comparison to microparticles of silica-functionalized magnetic particles. Such buffers accompanied by Fe 3 O 4 /SiO 2 nanoparticles segregate EBV and HBV to detect virus based upon amplification of PCR regarding two-fifty basepair fragments of "nuclear antigen encoding gene" particular to EBV and four-thirty-four basepair gene fragments particularly to HBV. Efficacy for purification of EBV and HBV employing synthesized nanoparticles came to be more desirable and valuable in comparison to microparticles of Fe 3 O 4 /SiO 2 , commercially available. The time necessary for the isolation of DNA consuming these nanoparticles had been considerably shortened because of the rapid attraction of nanoparticles towards magnets within fifteen to twenty seconds. While microparticles take 2-3 minutes. Supplementary experimentations engaging Fe 3 O 4 /SiO 2 nanoparticles for isolating nucleic acid out from other viral pathogens present inside blood samples are underway[START_REF] Quy | Synthesis of Silica-Coated Magnetic Nanoparticles and Application in the Detection of Pathogenic Viruses[END_REF].

Fig. 8 .

 8 Fig. 8. Process of extracting viral RNA through lysis, binding, washing and elution of samples

  laborious and inefficient operations of these methods. To resolve this problem, pcMNPs were synthesized by functionalizing MNPs with pc (poly (amino ester) bonded to carboxyl groups) for designing method of extraction of RNA based on pcMNPs meant for sensitive identification of SARS-CoV-2, triggering virus for COVID-19 as shown in Fig. 9. This technique involves lysis as well as bonding stages in one step. Prepared complex of pcMNPs-RNA were immediately inserted into successive RT-PCR reactions. The basic route purified viral RNA from compound samples in 20 minutes exploiting uncomplicated manual way, else automatic upgraded methodology. Through recognizing 2 dissimilar sections (N gene and ORFlab) in RNA of virus, 10-copy sensitivity along with strong relation ranging from 10 to 100,000 copies referring to corona pseudo-viral particles were attained. Advancing from the easiness and exceptional executions, this firsthand method of extraction strongly decreases the restoring time and functioning requirements in existing molecular diagnosis of COVID-19, particularly for the primary clinical diagnostics [94].

Fig. 9 .

 9 Fig. 9. Schematic of pcMNPs synthesized by functionalization of MNPs with pc (Poly (amino

Fig. 10 .

 10 Fig. 10. Schematic representation of the MNP synthesis. a) Synthesis of amino-magnetic

Fig. 11 .

 11 Fig. 11. a) Schematic of functionalized MNPs, b) schematic of mimic SARS-CoV-2, and c)

  Conventional diagnosis and detection approaches typically (i) need a long duration between collection of sample and result interpretation, (ii) have low sensitivity (iii) experimental processes are difficult, (iv) lack specificity, and (v) may have high false-negative rates. However, nanoparticles-based tools have the potential to overcome these limitations. They play an important role in rapid and sensitive diagnosis of several viral infections that allow early treatment and monitoring of positively identified patients, therefore saving suffering and lives.The application of MNPs for diagnosis has seen some positive examples including viralextraction and detection. These magnetic nanomaterials are the results of development made in the production and characterization of these materials in addition to appropriate functionalization. The opportunity to have diverse surface chemistry to provide functional groups and surface ligands on these MNPs makes these nanomaterials directly relevant for viraldiagnosis. The combination of extraction, enrichment, and detection permits the lowering of detection limit expressively. Magnetic nanoparticles for viral extraction to detection characterize a significant alternative involving progressing exploration. MNPs enable development of analytical devices that can recognize biologically selective substances present inside complex matrix sample with no long treatments of samples. These devices produce highly specific responses in the form of signals that are produced due to biological recognition using MNPs[START_REF] Campos | Recent Developments in Nanotechnology for Detection and Control of Aedes aegypti-Borne Diseases[END_REF].Despite countless benefits possessed by MNPs in comparison with orthodox techniques, research about toxicities as well as potentially harmful nanoparticle impact is further necessary.

Table of Contents

 of 1. Introduction ................................................................................................................................. 4 2. Limitations of conventional systems………….……………………………………….……….7 3. Non-magnetic nanoparticles in viral diagnosis ……………………………………...………..11 4. Contribution of magnetic nanoparticles (MNPs) in the diagnosis of viruses ........................... 16

	4.1. Extraction and purification of nucleic acid………….……………………………………….20
	4.2. Enrichment/concentration of target substance …………………….……………………….23
	4.3. Detection of nucleic acid……………………………………………………………………26
	5. Magnetic nanoparticles in diagnosis of coronavirus (COVID-19) ………………....……….32
	6. Conclusions and perspectives…………………………………………………………………39

Table 4

 4 Characteristics of few magnetic nanoparticles used in viral diagnosis.

	Particles		Size (nm)	Composition	Sensitivity	Ref
	characteristics
	Cross-Linked Iron	30	5 nm core with	Low nM∼pM,	[72]
	oxide			10nm	dextran	50 viruses/100
				coating		μL
	Iron Oxide		19.5			<1 nM	[73]
	Cubic	FeCo	12.8	1.5 nm oxidized	2 × 10 6 nucleic	[74]
	nanoparticles		shell		acids
	Antiferromagnetic	100	Multilayers of the	10 pM	[75]
	nanoparticles		ferromagnetic,
				interlayer	of
				nonmagnetic
				material
	γF 2 O 3		324±7	poly(styrene/acryla	2 × 10 -2 viruses	[76]
				mide)		/mL
	Fe 3 O 4 @SiO 2		-	APTES	(3-	4.42 × 10 -14 g/m	[77, 78]
				aminopropyl)trieth	L
				oxysilane) and SA

Acknowledgment

The authors would like to thank all those scientists whose research contributions in the field of viral diagnosis have been referred here. Supported by Institute of Analytical Sciences (ISA), University Claude Bernard Lyon-1, CNRS, ISA-UMR 5280, F-69622 Lyon, France.

increase the use of magnetic nanomaterials in management the COVID-19 outbreak and future epidemics.

Credit contribution statement

Sumera Khizar: Writing and editing review. Abdelhamid Elaissari: Editing and finalizing review. All authors have read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Alexandre Corazza
email: alexandre.corazza@creatis.insa-lyon.fr

Pauline Muleki-Seya
email: pauline.muleki-seya@creatis.insa-lyon.fr

Arthur Chavignon
email: arthur.chavignon@sorbonne-universite.fr

Olivier Couture
email: olivier.couture@sorbonne-universite.fr

Adrian Basarab
email: adrian.basarab@creatis.insa-lyon.fr

Barbara Nicolas
email: barbara.nicolas@creatis.insa-lyon.fr

Adaptive beamforming combined with decision theory-based detection for ultrasound localization microscopy

Abstract-Ultrasound Localisation Microscopy (ULM) is an imaging framework which consists in tracking microbubbles (MBs) on ultrasound (US) images to estimate their trajectory and thus the map of the vascular network. ULM algorithms takes as input US images usually beamformed with the delay-and-sum (DAS) method. In a previous study, we have shown that adaptive beamforming enhances the results of ULM on simulated data by detecting more MBs and localizing them more precisely. In another study, we introduced a new MB detection method based on decision theory. In this paper, adaptive beamformers such as Capon, pDAS and iMAP combined to this detection method are applied on in vivo rat brain data. Results show that this combination allow to identify more MBs and thus to represent more vessels in the ULM vascular network map.

Index Terms-Ultrasound Super-Resolution, adaptive beamforming, microbubble identification, decision theory

I. INTRODUCTION

ULM is a super-resolution imaging method providing information on vascular network structures and flow velocity by detecting, localising and tracking microbubbles (MBs). Its interest has been validated on rat [START_REF] Couture | Ultrasound Localization Microscopy and Super-Resolution: A State of the Art[END_REF] and human [START_REF] Demené | Transcranial ultrafast ultrasound localization microscopy of brain vasculature in patients[END_REF] brain as part of the study of stroke and brain diseases. It has also been applied to detect tumors [3] [4] and to monitor anticancer therapy [5] [6]. An example of ULM framework is described on Fig. 1. First, a liquid solution containing MBs is injected by vein (Fig. 1a). While the MBs are moving in the vascular network, carried away by the blood flow, US signals are acquired over time (Fig. 1b). These signals are beamformed to obtain US images (Fig. 1c). The tissue is filtered (Fig. 1d), generally with a singular value decomposition (SVD) [START_REF] Demené | Spatiotemporal Clutter Filtering of Ultrafast Ultrasound Data Highly Increases Doppler and fUltrasound Sensitivity[END_REF], in order to facilitate the MBs identification (Fig. 1e). The centroid of the identified MBs are localized with a precision beyond the diffraction limit (Fig. 1f). Finally, the centroid coordinates at different times are paired to track each MB (Fig. 1g) to build the vascular network map by accumulating the MBs trajectories (Fig. 1h). A prerequisite for ULM is to acquire ultrasound (US) images during a sufficient time such that the MBs can move through a maximum of vessels. Thus, there is a trade-off between acquisition time and the completion of the vascular network map. To deal with this trade-off, adaptive beamforming can be a solution. Indeed, the properties of US images could have a significant influence on ULM outputs. For instance, the minimum variance beamformer provides a better resolved point spead function (PSF) [START_REF] Asl | Contrast enhancement and robustness improvement of adaptive ultrasound imaging using forward-backward minimum variance beamforming[END_REF]. Other beamforming methods such as pDAS [START_REF] Polichetti | A nonlinear beamformer based on p-th root compression-application to plane wave ultrasound imaging[END_REF] or iMAP [START_REF] Chernyakova | IMAP Beamforming for High-Quality High Frame Rate Imaging[END_REF] highlight the MBs and attenuate the speckle and noise components on the US images. We studied the effect of these beamformers in [START_REF] Corazza | Microbubble detection with adaptive beamforming for ultrasound localization microscopy[END_REF] and showed that adaptive beamformers enhance the localisation precision and the detection rate on a simulated dataset. However, the benefits on in vivo studies were less evident. In addition to this study, we also introduced a new MB identification method based on decision theory [START_REF] Corazza | Microbubble identification based on decision theory for ultrasound localization microscopy[END_REF]. In this paper, we are pursuing our work by combining adaptive beamforming with this MB identification method and evaluate the results on an experimental rat brain dataset.

II. MATERIAL AND METHOD

A. In vivo dataset

The in vivo dataset is provided by the Performance Assessment of Localization Algorithms (PALA) toolbox [START_REF] Heiles | Performance benchmarking of microbubble-localization algorithms for ultrasound localization microscopy[END_REF], is available at https://zenodo.org/record/7883227. The RF data are acquired with a linear US probe emitting at 15 MHz (L22-14v, Verasonics, USA) and an US research scanner (Vantage 256, Verasonics, USA) on a rat brain during MB injections of 50 µL every 30 seconds. The acquisition process lasted 3 minutes and 20 seconds with a frame rate of 1000 Hz, with 3 plane waves tilted at -7 • , 0 • and 7 • angles.

B. Beamforming methods

Each scatterer that backscatters an echo from the plane wave excitation provides information about its echogenicity through the energy along the echo wave front. The first step of beamforming is to isolate this wave front by rephasing the received signals. The principle is based on time-arrival delay of an echo coming from coordinates (x, z) on each piezoelectric sensor along the linear probe. The second step is to sum the rephased signals, eventually weighted by a vector w manually set, to extract the pixel intensity at the coordinates (x, z). This method is called Delay-And-Sum (DAS) [START_REF] Perrot | So you think you can das? a viewpoint on delay-and-sum beamforming[END_REF]. The difference with adaptive beamforming is the weighting vector w that is adaptive, i.e. computed from the rephased signals in different ways depending on the beamforming method:

• the minimum variance, also known as Capon, is based on the rephased signal variance minimization to attenuate eventual disturbing sources [15] [8]. In practice, this strategy provides a thinner PSF, • the pDAS method [START_REF] Polichetti | A nonlinear beamformer based on p-th root compression-application to plane wave ultrasound imaging[END_REF] computes the weighting vector through an auto-correlation approximation of the rephased signal. In practice, this calculation rule leads to an attenuation of uncorrelated signal component such as noise and speckle. A parameter p defines the autocorrelation order. The method is referred to as 2pDAS for p = 2, • the iMAP beamformer [START_REF] Chernyakova | IMAP Beamforming for High-Quality High Frame Rate Imaging[END_REF] models the rephased signal as the sum of a constant component corresponding to the backscattered amplitude and a random Gaussian noise. By applying a maximum a posteriori strategy, the mean and standard deviation of a Gaussian distribution that maximize the similarity with the distribution of the rephased vector are estimated to isolate the constant component. In practice, the noise and speckle are attenuated. To estimate the mean and variance, the algorithm iterates through a loop. The method is referred to as 2iMAP for 2 iterations.

C. Microbubble identification methods

In the literature, three MB identification methods can be encountered. The first, based on the intensity, consists in finding the local maximas higher than a threshold empirically chosen [START_REF] O'reilly | A super-resolution ultrasound method for brain vascular mapping[END_REF] [START_REF] Ackermann | Detection and tracking of multiple microbubbles in ultrasound B-mode images[END_REF]. An equivalent method is to set a number N of MBs to detect per image and to select only the N higher local maximas [START_REF] Couture | Ultrasound Localization Microscopy and Super-Resolution: A State of the Art[END_REF]. However, some MBs intensities are lower than noise. Thus, the second identification method is intensityindependant and is based on normalized cross-correlation (NCC) [START_REF] Bourquin | In vivo pulsatility measurement of cerebral microcirculation in rodents using dynamic ultrasound localization microscopy[END_REF] [START_REF] Song | Improved super-resolution ultrasound microvessel imaging with spatiotemporal nonlocal means filtering and bipartite graph-based microbubble tracking[END_REF]. The aim of this method is to detect structures similar in shape to a MB model, often estimated as a Gaussian shape [START_REF] Song | Improved super-resolution ultrasound microvessel imaging with spatiotemporal nonlocal means filtering and bipartite graph-based microbubble tracking[END_REF], with simulation [START_REF] Bourquin | In vivo pulsatility measurement of cerebral microcirculation in rodents using dynamic ultrasound localization microscopy[END_REF] or experimentally by imaging a wire cross-section [START_REF] Christensen-Jeffries | In vivo acoustic super-resolution and super-resolved velocity mapping using microbubbles[END_REF] [START_REF] Christensen-Jeffries | Microbubble Axial Localization Errors in Ultrasound Super-Resolution Imaging[END_REF]. A threshold τ is applied on the NCC results to extract the structures with the highest similarities compared to the MB model. We proposed a third method based on decision theory (DT) with the Neyman-Pearson criterion [START_REF] Corazza | Microbubble identification based on decision theory for ultrasound localization microscopy[END_REF], which is independant on intensity and PSF shape. Its principle is to study each pixel along the time dimension. The temporal signal for one pixel is modeled as the sum of a Gaussian noise induced by tissue movement and electronical noise and a constant component corresponding to a MB passage on the pixel. The decision theory tools allow to compute an adaptive threshold different for each pixel in order to isolate the constant component with a false alarm rate α 0 set by the user. It provided a more complete and better-resolved vessel map [START_REF] Corazza | Microbubble identification based on decision theory for ultrasound localization microscopy[END_REF].

D. Ultrasound Localization Microscopy method and beamforming

The ULM framework used is available on the GitHub page of the PALA toolbox https://github.com/AChavignon/PALA related to the work in [START_REF] Heiles | Performance benchmarking of microbubble-localization algorithms for ultrasound localization microscopy[END_REF]. The NCC and decision theory based identification methods can be added to this toolbox with the code available on the GitHub page https://github.com/CorazzaAlexandre/Microbubble detection. The beamforming methods are also available at https://github.com/CorazzaAlexandre/PALA Beamforming. The ULM parameters used are summarized in the Table I. Note that the SVD filter is applied on the RF data, before beamforming since pDAS and iMAP can highlight tissue components and make it difficult to filter after beamforming. To limit the computation time of this step, we used the randomized SVD [START_REF] Song | Accelerated Singular Value-Based Ultrasound Blood Flow Clutter Filtering with Randomized Singular Value Decomposition and Randomized Spatial Downsampling[END_REF]. Note that intensity-based MB identification is not suitable due to MB slight attenuation with pDAS or iMAP. Either the NCC identification is not used due to its inability to link some vessels as highlighted in [START_REF] Corazza | Microbubble identification based on decision theory for ultrasound localization microscopy[END_REF].

III. RESULTS AND DISCUSSION

The beamformers are applied to the in vivo rat brain RF signals after SVD filtering. An example for each beamforming method is given in Fig. 2. It can be seen by taking the DAS as reference that Capon reduces the PSF size, and that 2pDAS and 2iMAP attenuate the speckle. The effect on the decision theory identification is studied through the threshold maps on Fig. 3. As expected, for DAS, some low threshold areas are drawn in the vessels where MBs are moving and high threshold in the tissue areas. With Capon, the threshold map is equivalent to DAS. However, with 2pDAS and particularly with 2iMAP, these low threshold areas are more resolved and a new one appears in the long vessels at the bottom of the image. There is a high MB concentration in these long vessels, and it has been discussed in [START_REF] Corazza | Microbubble identification based on decision theory for ultrasound localization microscopy[END_REF] that this could be a limit for the decision theory method. Indeed, frequent passage of bubbles over a pixel biases the estimation of the noise mean and standard deviation respectively by the median and the median absolute deviation (MAD). Thus, it is interesting to see that adaptive beamforming is able to overcome this limit. An hypothesis is that 2pDAS and 2iMAP enhance the signal-tonoise ratio (SNR) which helps to distinguish noise from MBs, and thus to correctly estimate the noise mean and standard deviation. The vacular network map provided by the ULM algorithm with DT identification for each beamforming methods are presented on the Fig. 5b andc indicate that the identified MBs lead to equivalent tracking qualities with each beamforming method. However, the resolution estimated by the Fourier Ring Correlation (FRC) [START_REF] Hingot | Measuring image resolution in Ultrasound Localization Microscopy[END_REF] is slighly lower with the adaptive beamforming passing from 13 µm with DAS to 13.9 µm with Capon and 15.1 µm with 2pDAS and 2iMAP.

Although FRC is robust to noise, this degradation in resolution can be caused by temporally correlated noise, i.e. undesirable temporally recurrent trajectories, e.g. the two artefacts at the top right and left corners. It is discussed in [START_REF] Hingot | Measuring image resolution in Ultrasound Localization Microscopy[END_REF] that these artifacts can bias the FRC. To conclude, although the images show areas with enhanced visibility (white arrows), there is currently no overall quantitative criterion for quantifying this improvement. Fig. 3: Threshold maps computed on 800 images (linear scale between 0 and 1). 2iMAP allow to identify and track more MBs. In perspective, it would be interesting to apply these adaptive beamformers on additional in vivo datasets to generalize these conclusions.

Fig. 1 :

 1 Fig. 1: ULM framework example.

4

 4

  (c) to (f). For visual comparison, the results with the intensity and NCC detections, both with DAS beamforming are shown on Fig. 4(a) and (b). The first map on Fig. 4(a) has a lack of detection and the second on Fig. 4(b) fails to connect some vessels between them (see areas Z i ). The maps obtained with the DT detection on Fig. 4(c) to (f) are quite similar, but some vessels indicated by the arrows are more filled with to 2pDAS and 2iMAP. To quantify these enhancement, different quantitative metrics are shown on Fig. 5. The saturation curves obtain with the different beamformers on Fig. 5a can be considered equivalent. The track length and velocity statistics on Fig.

Fig. 2 :

 2 Fig. 2: Rat brain US images with different beamformers (log scale, dynamic of 60dB).

Fig. 4 :Fig. 5 :

 45 Fig. 4: ULM results with different beamformers.

TABLE I :

 I ULM parameters.

	Parameter	Value
	Tissue filtering	SVD (cutoff: 10)
	Beamforming	DAS, Capon, pDAS or iMAP
	US image pixel size	λ = 100µm
		Intensity (N=100 MBs per images)
	MB identification	NCC (Gaussian PSF model, τ = 0.6)
		DT (α 0 = 0.1%)
	ROI size	5λ
	Isolation	3 local maximas per ROI maximum
	Localisation	Radial symmetry based method [23]
		Hungarian algorithm
	Tracking	max linking distance: 2λ,
		minimal track length: 10 images
	Density map pixel size	λ/10

ACKNOWLEDGMENTS

This work was supported by the LABEX CELYA (ANR-10-LABX-0060) and LABEX PRIMES (ANR-11-LABX-0063) of Université de Lyon, within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). This work was partly funded by France Life Imaging (grant ANR-11-INBS-0006).