\r\n\tInitially, macroecology includes the study of food and space of species of particular area but now it encompasses the relationships between organisms and environment characterized by their distribution, diversity and patterns of abundance. The spatial macroecological investigations are important and being carried out on the dynamics of functional ecosystem and climate change, biodiversity and fisheries. \r\n\t \r\n\tThis book covers the findings and reviews of various aspects of freshwater ecosystems which highlight not only the underlying challenge of macroecological processes but also the emerging traits of functional diversity including micro-organisms, algae, macrophytes, invertebrates, fish and other vertebrates. \r\n\t \r\n\tThe book will be useful for those who are working in the field of freshwater ecosystems. Each chapter of the book focuses on the basic and current status of the knowledge based on the concerned topics in the field of freshwater ecology. Hopefully the book will be beneficial not only for academicians but also for \r\n\tindustrialists and professionals.
",isbn:null,printIsbn:"979-953-307-X-X",pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,isSalesforceBook:!1,isNomenclature:!1,hash:"da20df3bd2e47c1599b87d037f4d74cb",bookSignature:"Dr. Mohammad Serajuddin",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8930.jpg",keywords:"Biodiversity, Running water, Aquatic Organisms, Anthropogenic Activities, Freshwater, Conservation, Strategies, Ecology, Flora, Alkalinity, Fish culture, Phytoplankton and Zooplankton",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"March 20th 2019",dateEndSecondStepPublish:"November 15th 2019",dateEndThirdStepPublish:"January 14th 2020",dateEndFourthStepPublish:"April 3rd 2020",dateEndFifthStepPublish:"June 2nd 2020",dateConfirmationOfParticipation:null,remainingDaysToSecondStep:"3 years",secondStepPassed:!0,areRegistrationsClosed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:null,coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"309081",title:"Dr.",name:"Mohammad",middleName:null,surname:"Serajuddin",slug:"mohammad-serajuddin",fullName:"Mohammad Serajuddin",profilePictureURL:"https://mts.intechopen.com/storage/users/296536/images/system/296536.jpeg",biography:"Dr. M. Serajuddin is presently a Professor in the Department of\nZoology, University of Lucknow, U.P., India. He is a Fellow of\nthe Zoological Society of India and Academy of Environmental\nBiology. He has received several awards of national and international reputes. He has been involved in research and teaching for\nover 25 years in fish biology, prevention of cancer through fish\nlipids, and fish biodiversity. Dr. Serajuddin acted as Principal\nInvestigator for major research projects funded by the Government of India. He has\nsupervised 15 Ph.D. students in zoology. He has to his credit more than 80 original\nresearch papers in peer-reviewed journals of national and international repute. He\nhas also edited 6 books bearing ISBN numbers.",institutionString:"University of Lucknow",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"University of Lucknow",institutionURL:null,country:{name:"India"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"5",title:"Agricultural and Biological Sciences",slug:"agricultural-and-biological-sciences"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"205697",firstName:"Kristina",lastName:"Kardum Cvitan",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/205697/images/5186_n.jpg",email:"kristina.k@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"6418",title:"Hyperspectral Imaging in Agriculture, Food and Environment",subtitle:null,isOpenForSubmission:!1,hash:"9005c36534a5dc065577a011aea13d4d",slug:"hyperspectral-imaging-in-agriculture-food-and-environment",bookSignature:"Alejandro Isabel Luna Maldonado, Humberto Rodríguez Fuentes and Juan Antonio Vidales Contreras",coverURL:"https://cdn.intechopen.com/books/images_new/6418.jpg",editedByType:"Edited by",editors:[{id:"105774",title:"Prof.",name:"Alejandro Isabel",surname:"Luna Maldonado",slug:"alejandro-isabel-luna-maldonado",fullName:"Alejandro Isabel Luna Maldonado"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"10359",title:"Landraces",subtitle:"Traditional Variety and Natural Breed",isOpenForSubmission:!1,hash:"0600836fb2c422f7b624363d1e854f68",slug:"landraces-traditional-variety-and-natural-breed",bookSignature:"Amr Elkelish",coverURL:"https://cdn.intechopen.com/books/images_new/10359.jpg",editedByType:"Edited by",editors:[{id:"231337",title:"Dr.",name:"Amr",surname:"Elkelish",slug:"amr-elkelish",fullName:"Amr Elkelish"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophile",surname:"Theophanides",slug:"theophile-theophanides",fullName:"Theophile Theophanides"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"314",title:"Regenerative Medicine and Tissue Engineering",subtitle:"Cells and Biomaterials",isOpenForSubmission:!1,hash:"bb67e80e480c86bb8315458012d65686",slug:"regenerative-medicine-and-tissue-engineering-cells-and-biomaterials",bookSignature:"Daniel Eberli",coverURL:"https://cdn.intechopen.com/books/images_new/314.jpg",editedByType:"Edited by",editors:[{id:"6495",title:"Dr.",name:"Daniel",surname:"Eberli",slug:"daniel-eberli",fullName:"Daniel Eberli"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"51557",title:"Influenza Inactive Virus Vaccine with the Fusion Peptide (rTα1- BP5) Enhances Protection Against Influenza Through Humoral and Cell-Mediated Immunity",doi:"10.5772/64403",slug:"influenza-inactive-virus-vaccine-with-the-fusion-peptide-rt-1-bp5-enhances-protection-against-influe",body:'\n
1. Introduction
\n
Avian influenza virus (AIV) is an enveloped virus that belongs to the Orthomyxoviridae family and has an eight-segmented, single-stranded, negative-sense RNA genome. Among the proteins encoded by the genome, there are two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) [1]. AIVs are classified into subtypes according to the combination of 16 HA and nine NA molecules. Among the many subtypes of AIV, H9N2 is thought to have originated from shorebirds and gulls, and has rapidly spread to become one of the most prevalent diseases in domestic poultry worldwide. It also causes serious economic loss in the poultry industry [2] (see Table 1).
In domestic avian species in North America, H9N2 influenza viruses occur primarily in turkeys, occasionally in quail, and rarely if ever in chickens. The H9N2 virus subtype was first isolated from turkeys in 1966 [3], when the virus was associated with mild respiratory disease. In Asia, long-term surveillance in live poultry markets in Hong Kong from 1975 to 1985 detected H9N2 influenza viruses in apparently healthy ducks but not in chickens [4]. Since the early 1990s, H9N2 influenza viruses have become widespread in domestic chickens in Asia [5]. Among the avian influenza A virus subtypes, H9N2 viruses have the potential to cause an influenza pandemic because they are widely prevalent in avian species in Asia and have demonstrated the ability to infect humans [6]. In April 1999, two World Health Organization reference laboratories independently confirmed the isolation of avian influenza A (H9N2) viruses for the first time in humans [7].
\n
The best protection against influenza virus infection remains effective vaccination [8]. Inactivated vaccines have been undergoing clinical trials as pandemic vaccine candidates, and it has been shown that inactivated vaccines elicit strong humoral responses; however, it is commonly accepted that no adequate mucosal or cellular immunity is achieved [9]. Adjuvants are able to improve the quantity and quality of innate immune responses by enhancing their speed and duration, and by inducing adequate adaptive immunity [10]. To improve methods for influenza vaccine production, the current strategy of many investigators is to increase the efficacy of pandemic influenza vaccines by the addition of adjuvants to boost immune responses, such as aluminum salts, MF59, IC31®, and chitosan [11–14].
\n
A defined peptide sequence able to stimulate specific immune cell subsets has the potential to act as an adjuvant for a variety of immunogens. The thymus is an important central immune organ for T-lymphocyte differentiation and maturation [15]. It is capable of secreting many peptides with the functions of regulating the development of different phenotypic markers and lymphocyte [16]. Thymosin alpha 1 (Tα1), an immunomodulatory peptide consisting of 28 amino acid residues, was isolated originally from calf thymus [17]. As a biological response modifier (BRM), Tα1 has multiple biological activities in the immune system. It can promote specific lymphocyte functions, stimulate the production of lymphokines such as gamma interferon (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin 2 (IL-2), macrophage migration inhibitory factor (MIF), and precursor stem cell into the CD4+/CD8+ T cells, increase T-cell proliferation, differentiation and maturation, and so on [18, 19]. Furthermore, it has the activities of antitumor and protection against oxidative damage [20]. Consequently, Tα1 is widely used in clinic treating various diseases including immunodeficiency diseases, severe sepsis, and systemic infectious disorder [21].
\n
The bursa of Fabricius (BF) is a primary humoral immune organ unique to birds and is the site of B-lymphocyte development and differentiation. The tripeptidebursin (LysHisGlyNH2) has been described as an endogenous B-cell stimulant or differentiation factor [22]. BS and bursin-like peptide T-X-N-L-K-H-G significantly enhance the JEV subtype vaccine-induced immune response in immunized mice [23]. Bursin-like epitope peptide (BLP) is one of bursin-like peptides and enhances immune responses in mice immunized with inactivated H9N2 avian influenza vaccine [24]. Our previous study has been reported that Bursopentin (BP5) is a small peptide separated from BF, which amino acid sequence is CKDVY. We found that BP5 not only promotes T-cell and B-cell proliferation, enhances humoral immunity and cellular immunity but also balances Th1 and Th2 immune responses [25, 26].
\n
Although both Tα1 and BP5 have the potent adjuvant effects, this study designed and synthesized Tα1-BP5 fusion gene according to the preferential codons of Escherichia coli, fused with prokaryotic expression vector pET-32a, and then transferred into E. coli BL21 to induce its expression. Then, we tested whether rTα1-BP5 could enhance immune responses in chicken upon vaccination with H9N2 avian influenza whole-inactivated virus (WIV).
\n
\n
2. Materials and methods
\n
2.1. Plasmid, viruses, and reagents
\n
pET-32a (+), E. coli DH5α, E. coli BL21 (DE3), pET32a (+)-BP5, and avian influenza virus A/Chicken/Jiangsu/JS-1/2002(H9N2) were maintained in our laboratory. Avian influenza virus A/Chicken/Jiangsu/NJ08/05(H9N2) was kindly provided by Dr. Qi-Sheng Zheng. Virus titers were determined in MDCK cells. H9N2 avian influenza whole-inactivated virus (WIV) was prepared by diluting the virus 1:4000 (v/v) in formalin [27, 28]. All restriction enzymes and Taq polymerase were purchased from TakaRa Biotechnology (Dalian, China). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (New York, NY, USA). Isopropyl-β-d-thiogalactoside (IPTG), 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT), TEMED, dNTP, Concanavalin A (ConA), Phorbol-12-myristate-13-acetate (PMA), and tetramethylbenzidine (TMB) were purchased from Jiu Shi Corporation (Zhengzhou, China). Horseradish peroxidase (HRP)-conjugated goat anti-mice IgG was obtained from Boshide Corporation (Wuhan, China). Control Standard Tα1 and BP5 peptides were synthesized by Shanghai Science Peptide Biological Technology Co., Ltd. (Shanghai, China), and the purity was over 95%.
\n
\n
2.2. Chicken embryos, animals, and vaccines
\n
Specific pathogen-free (SPF) Roman chicken and chicken embryos were obtained from the Henan Experimental Animal Research Center. Avian influenza virus A/Chicken/Jiangsu/NJ08/05(H9N2) (107 TCID50/0.1 mL) was inoculated into the allantoic cavities of 10-day-old SPF chicken embryos; the embryos that died within 24 h were discarded, and the allantoic fluids were harvested from the infected embryos at 48 h postinfection and inactivated by treatment with 0.2% formalin. The inactivated virus was emulsified with mineral oil to make an oil-formulated inactivated H9N2 AIV vaccine. One dose of the vaccine contained 107 TCID50/0.1 mL, which was equal to it before inactivation. Procedure and test of inactivated vaccine were described according to OIE Terrestrial Manual 2012 [29].
\n
\n
2.3. Gene cloning and expression of the recombinant fusion peptide Tα1-BP5
\n
Gene of the recombinant fusion peptide thymosin α1-Bursopentin (Tα1-BP5) was designed according to the preferential codons of E. coli and amplified by splicing overlap extension polymerase chain reaction (SOE-PCR) method [30]. Sequences of the primers used for the synthetic Tα1-BP5 are as follows: F1: 5′-CCG GAA TTC AGC GAC GCT GCT GTT GAC ACT AGC AGC GAA ATC ACT ACTA AAG ACT TG-3′; F2: 5′-GTT CGG GGT GCTG CCG CCG CCG CCG TTT TCA GCT TCT TCA ACA ACT TCT TTT TTT TCT TTC AAG TCT TTA GTA GT-3′; and F3: 5′-GGC GGC GGC GGC AGC TGC AAA AAT GTG TAT TAA GTC GAC TCG-3′, with EcoR I and SaI I site (underlined). The genes of Tα1 and BP5 were connected with the gene of the GGGGS linker to form the Tα1-BP5 fusion gene. And then, the amplified DNA fragment was digested by EcoR I and SaI I, and then ligated into the expression vector pET32a. The ligation mix was transformed into competent DH5α cells and the single bacterial colony was selected by overnight growth on Luria broth (LB) agar plates containing 100-μg/mL ampicillin. The obtained recombinant plasmid pET32a-Tα1-BP5 was confirmed by restriction endonuclease digestion and DNA sequencing. The pET32a-Tα1-BP5 plasmid was transformed into E. coli BL21 (DE3) for inducing expression. The expression products were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). And the recombinant proteins were purified on a Ni-affinity chromatography column (Amersham Bioscience HiTrap chelating HP 5 mL × one column) following the manufacturer’s instructions.
\n
\n
2.4. Activity testing of fusion peptide Tα1-BP5 (rTα1-BP5) in vitro
\n
Thymus and spleens from 4 to 6-week-BALB/c mice with (20 ± 2) g were collected aseptically, put them at 200-mesh stainless screen mesh cells, and gently minced into single cell suspension with a syringe followed by adding Hank’s solution. The red blood cells were removed by centrifugation at 500 rpm for 5 min. The supernatant was centrifuged at 500 rpm for 5 min. The obtained pellet was washed with Hank’s solution twice. The density of lymphocytes was adjusted to around 5 × 106 cells/mL using RPMI-1640 medium containing 10% FBS. ConA and PMA were added into thymic lymphocytes and splenic lymphocytes to make the concentrations reach 5 μg/mL and 300 ng/mL, respectively. The two kinds of solutions were subpackaged into a 96-well plate with 100 μL/well, respectively, and three parallel samples were set for each well. The plates were incubated in CO2 incubator at 37°C for 6 h, followed by adding 100-μL/well rTα1-BP5 (affinity chromatography purified through Ni column) with different concentrations (1.25, 2.5, 5.0, 10.0, and 20.0 µg/mL) and continued culturing for 72 h. Control groups (phosphate-buffered saline (PBS), 10.0 µg/mL thioredoxin, 10.0 µg/mL Tα1, and 10.0 µg/mL BP5) were used following the same procedures. MTT method was used to test the effect of rTα1-BP5 effect on thymic and splenic lymphocytes proliferation. Relative ratio of cell proliferation (%) = (experimental group OD570/control group OD570) × 100% [31, 32].
\n
\n
2.5. Immunization of chickens
\n
All animal experiments were approved by the Henan University of Science and Technology Animal Care and Use Committee. Twenty-one-day-old SPF Roman chickens were randomly divided into six experimental groups of 25 chickens each and intramuscularly immunized two times on days 0 and 14 with (i) 100 μL PBS as a negative control, (ii) 100 μL H9N2 WIV (A/Chicken/Jiangsu/NJ08/05, 107 TCID50/0.1 mL), (iii) a mixture of 100 μL H9N2 WIV and Tα1 (50 μg), (iv) a mixture of 100 μL H9N2 WIV and BP5 (50 μg), (v) a mixture of 100 μL H9N2 WIV and rTα1-BP5 (50 μg), and (vi) 100 μL oil-formulated inactivated H9N2 AIV vaccine (A/Chicken/Jiangsu/NJ08/05, 107 TCID50/0.1 mL) as a positive control (Table 2).
\n
Figure 1.
Experimental scheme of immunization, sample collection, and challenge.
\n
\n
\n
\n\n
\n
Group
\n
Vaccination on days 0 and 14a
\n
\n\n\n
\n
1
\n
100 μL PBS
\n
\n
\n
2
\n
107 TCID50 H9N2 WIV
\n
\n
\n
3
\n
107 TCID50 H9N2 WIV + 50 μg Tα1
\n
\n
\n
4
\n
107 TCID50 H9N2 WIV + 50 μg BP5
\n
\n
\n
5
\n
107 TCID50 H9N2 WIV + 50 μg rTα1-BP5
\n
\n
\n
6
\n
107 TCID50 H9N2 AIV vaccine
\n
\n\n
Table 2.
Animal groups and the experimental design.
a H9N2 WIV, inactivated H9N2 avian influenza whole-inactivated virus; H9N2 AIV vaccine, H9N2 avian influenza virus vaccine prepared with oil/water as an adjuvant.
\n
The details of the animal experiment time points are shown in Figure 1.
\n
\n
2.6. Detection of antibodies in serum
\n
Chicken (n = 5 per group) sera were collected on 7 and 21 days after the first immunization. Serum antibody (HI and antigen-specific antibodies) titers were determined using standard HI microliter and enzyme-linked immunosorbent assay (ELISA) as described [25, 33]. Briefly, to detect the HI titers in chicken serum, sera were inactivated by incubation for 30 min at 56°C and serially diluted twofold in PBS, then transferred in duplicate to 96-well round-bottomed plates. Standard avian influenza virus (A/Chicken/Shandong/6/96(H9N2)) antigen with four units was then added to each diluted serum samples in a volume of 50 μL, and followed by an equal volume of 0.5% chicken erythrocyte suspensions. The mixture was incubated for 1 h at room temperature before the results were read. The HI titers were defined as the highest serum dilution capable of preventing hemagglutination.
\n
To evaluate the antigen-specific antibodies titers, ELISA plates were coated with 10-µg/mL recombinant influenza HA protein (expressed in E. coli BL21) and blocked with 1% bovine serum albumin (BSA) for 2 h at 37°C. Aliquots of diluted chicken sera were added to the plates, which were then incubated overnight, washed, and incubated with HRP-conjugated goat anti-chicken IgG. Finally, TMB was added and the reaction was stopped by the addition of 2N H2SO4 and the absorbance was read at OD450. Each serum sample was repeated in quintuplicate. The results were plotted as OD versus dilution (log scale). Titers at half maximal OD were determined by linear interpolation [34].
\n
\n
2.7. Determination of AIV-neutralizing antibodies
\n
Inactivated sera were incubated with 100 plaque-forming unit (pfu) of avian influenza virus (A/Chicken/Jiangsu/JS-1/2002(H9N2), and the titers of AIV-neutralizing antibodies determined as described [35].
\n
\n
2.8. Cytokine assays
\n
On 7 and 21 days after the first immunization, the serum levels of Th1-type cytokine (IFN-γ) in chickens were determined using commercial Chicken cytokines gamma interferon ELISA kits (Cusabio Biotech, MD, USA), whereas Th2-type cytokine (IL-4) was determined with another commercial Chicken cytokines interleukin 4 ELISA kits (Cusabio Biotech, USA). The procedure followed the manufacturer’s instructions.
\n
\n
2.9. Lymphocyte proliferation response
\n
To detect changes in cellular immunity, lymphocyte proliferation response was performed. Thymus and bursa of Fabricius were collected from immunized chickens at 7 and 21 days after the first immunization. The thymus and BF lymphocytes were isolated and maintained in 1640 medium supplemented with 10% FBS at 37°C with 5% CO2. The thymus lymphocytes (5 × 106 cells/mL) were seeded in a 96-well plate and incubated with 50 μL of ConA (40 μg/mL) at 40°C/5% CO2 for 48 h, whereas the BF lymphocytes (5 × 106 cells/mL) were treated with 50 μL of PMA (1 μg/mL) in a 96-well plate at 40°C/5% CO2 for 24 h. Then, the lymphocyte proliferation assay was performed using a standard MTT method as described previously [36, 37]. Then, the plate was incubated with 10 μL of 5 mg/mL MTT for 3 h. Finally, 100 μL of 10% (w/v) SDS in 0.01 M HCl was added into the plate and allowed to incubate for 2 h. A spectrophotometric measurement was taken at A570.
\n
\n
2.10. Virus challenge experiment
\n
Two weeks after the second vaccination, chickens (n = 15 per group) were intranasally challenged with 2.5 × 106 TCID50 avian influenza virus A/chicken/Jiangsu/JS-1/2002(H9N2) in 100 μL PBS. Five chickens per group were humanely sacrificed at 3, 5, and 7 days after virus challenge and the viral titers in their lungs assessed by plaque formation assays using MDCK cells as described [35].
\n
\n
2.11. Statistical analysis
\n
Statistical analyses were performed using unpaired t-tests or one-way analysis of variance (ANOVA) F-statistics followed by GraphPad Prism 6 software. Data are presented as the mean ± standard deviation (SD). Turkey multiple comparison tests were used to assess differences among the five experimental groups, with differences being considered significant at P < 0.05 or P < 0.01.
\n
\n
\n
3. Results
\n
3.1. Expression of the recombinant fusion peptide Tα1-BP5
\n
The gene of Tα1-BP5 was amplified by SOE-PCR with the primers F1, F2, and F3. The PCR products were identified by electrophoresis, and then about 114bp strip was observed. The recombinant plasmid was extracted and identified with Hind III enzyme. The results showed that the recombinant plasmid was not digested by Hind III, indicating that the recombinant plasmid had deleted the Hind III restriction site (Figure 2A). Sequencing result showed that the gene of the recombinant fusion peptide Tα1-BP5 was inserted into pET32a vector, and it was consistent to the expected size (Table 3), which was suggested that the recombinant expression vector was constructed successfully, and it was named pET32a-Tα1-BP5. Then, pET32a-Tα1-BP5 was transferred into E. coli BL21 (DE3) for its expression, and the expressed products were detected by using SDS-PAGE. The result showed that Tα1-BP5 was expressed and purified with the molecular weight of 31.4 kDa (Figure 2B), which is consistent with its predicted molecular weight.
\n
\n
\n
\n\n
\n
Name
\n
Amino acid sequence
\n
\n\n\n
\n
Tα 1-BP5
\n
Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys Asp Leu Lys Glu Lys Lys Glu Val Val Glu Glu Ala Glu Asn Gly Gly Gly Gly Ser Cys Lys Asp Val Tyr
\n
\n\n
Table 3.
Amino acid sequence of recombinant fusion peptide Tα1-BP5.
\n
Figure 2.
Identification of the recombinant plasmid pET32a-Tα1-BP5 and the expression of fusion peptide Tα1-BP5 in E. coli. (A) 1: DL2000 marker; 2: the recombinant plasmid pET32a-Tα1-BP5 after Hind III enzyme digestion; and 3: the recombinant plasmid pET32a-Tα1-BP5. (B) 1: low molecular weight protein marker; 2: not induced E. coli BL21 (DE3); 3: Not induced recombinant E. coli BL21 (DE3)/pET32a-Tα1-BP5; 4: induced recombinant E. coli BL21 (DE3)/pET32a-Tα1-BP5.
\n
\n
3.2. Activity of rTα1-BP5 in vitro
\n
The expressed product of TBP5 recombinant bacteria was affinity chromatography purified through protein Ni column and quantified through spectrophotometer. MTT method was used to test the effect of rTα1-BP5 on the proliferation of mouse thymic and splenic lymphocytes. The results showed that all rTα1-BP5 with different concentrations (1.25, 2.5, 5.0, 10.0, and 20.0 µg/mL) could promote the proliferation of thymic and splenic lymphocytes compared to PBS group. rTα1-BP5 could stimulate thymic and splenic lymphocytes proliferation stronger than TP5 and BP5. The differences were significant (P < 0.05) on the concentrations of 5.0 and 20.0 µg/mL, and the differences were more significant (P < 0.01) on the concentrations of 10.0 µg/mL (Figure 3A and B). All these data demonstrated that rTα1-BP5 could promote the proliferation of mouse thymic T lymphocytes and splenic B lymphocytes.
\n
Figure 3.
The effects of rTα1-BP5 on the proliferation of thymic lymphocytes (A) or splenic lymphocytes (B) from immunized mice. The data presented are of five replicates. *, P < 0.05, compared with mice immunized with PBS, and **, P < 0.01, compared with mice immunized with BP5 or Tα1.
\n
Figure 4.
Effect of rTα1-BP5 to H9N2 AIV vaccination on antigen-specific HI titers and anti-HA IgG antibodies. Chickens were immunized two times, and chicken sera were collected on days 7 and 21 after the first immunization, and the serum HI titers (A) and IgG titers (B) were analyzed by HI assay and ELISA, respectively. The data presented are means ± SD of results from five replicates. *, P < 0.05, and **, P < 0.01, compared with chickens immunized with H9N2 WIV alone.
To determine antigen-specific immune responses to immunization, chickens were immunized two times, then sera were taken on days 7 and 21 after the first immunization and detected for HI and anti-HA antibody titers. HI antibody titers of chickens immunized with inactivated vaccine, Tα1 combined with H9N2 WIV and BP5 combined with H9N2 WIV increased significantly compared with chickens immunized with the H9N2 WIV alone at days 7 and 21 (P < 0.05). However, HI antibody titers in chickens immunized with rTα1-BP5 combined with H9N2 WIV were significantly higher than in chicken immunized with BP5 combined with H9N2 WIV at days 7 and 21 (P < 0.05) (Figure 4A). Anti-HA IgG antibody was observed in immunized chickens on days 7 and 21 after the first immunization. rTα1-BP5 enhanced the secretion of IgG antibody on day 7 after the first immunization, and the effect was greater than that induced by H9N2 AIV vaccine, Tα1 combined with H9N2 WIV and BP5 combined with H9N2 WIV. On day 21 after the first immunization, BP5 significantly enhanced IgG antibody secretion levels compared with that induced by H9N2 vaccine, Tα1 combined with H9N2 WIV (P < 0.05), while the effect of rTα1-BP5 was the greatest than the other groups (P < 0.01) (Figure 4B). These results suggested that rTα1-BP5 stimulates significant antigen-specific immune responses.
\n
\n
3.4. rTα1-BP5 promoted the production of AIV-neutralizing antibody
\n
To assess whether rTα1-BP5 can effectively enhance virus-neutralizing antibodies, chicken sera were collected on days 7 and 21 after the first immunization and the titers of AIV-neutralizing antibody were assessed. The result showed that the titers of neutralizing antibody of chickens immunized with Tα1 plus H9N2 WIV, BP5 plus H9N2 WIV, and H9N2 AIV vaccine were higher than that in chickens immunized with H9N2 WIV alone on day 7, while it was higher in chickens immunized with Tα1-BP5 plus H9N2 WIV than that of other groups. Consistent with this, AIV-neutralizing antibody titers of chicken injected with rTα1-BP5 plus H9N2 WIV were the highest on day 21 (Table 4). These results indicated that rTα1-BP5 significantly stimulates the production of AIV-neutralizing antibodies.
\n
\n
\n
\n
\n\n
\n
Treatment
\n
PRNT50a
\n
\n
\n
First boost
\n
Second boost
\n
\n\n\n
\n
PBS
\n
–
\n
–
\n
\n
\n
H9N2 WIV
\n
10 ± 0.38
\n
19 ± 0.21
\n
\n
\n
H9N2 WIV + Tα1
\n
15 ± 0.30*
\n
22 ± 0.25*
\n
\n
\n
H9N2 WIV + BP5
\n
18 ± 0.32*
\n
28 ± 0.43*
\n
\n
\n
H9N2 WIV + rTα1-BP5
\n
22 ± 0.24**
\n
32 ± 0.19**
\n
\n
\n
Inactivated H9N2 AIV vaccine
\n
16 ± 0.15*
\n
25 ± 0.54**
\n
\n\n
Table 4.
Titers of plaque-reducing neutralizing antibody in groups of chicken.
a Chickens were vaccinated on days 0 (first boost) and 14 (second boost). Chicken sera (n = 5) were collected on days 7 and 21, and plaque-reducing neutralizing antibody titers were determined. The 50% plaque-reducing neutralizing titer (PRNT50) was reported as the geometrical reciprocal of the serum dilution resulting in a 50% reduction in plaques. The data presented are means ± SD of results from five replicates.
*, P < 0.05, and
**, P < 0.01, compared with chickens immunized with H9N2 WIV alone.
\n
\n
3.5. rTα1-BP5 increases the production of both Th1- and Th2-type cytokines
\n
We then examined the levels of Th1 (IFN-γ) and Th2 (IL-4) cytokines from immunized chickens. Compared with stimulation with H9N2 WIV alone, both IFN-γ and IL-4 secretion were remarkably increased after immunization with inactivated H9N2 AIV vaccine, Tα1 plus H9N2 WIV and BP5 plus H9N2 WIV at days 7 and 21, and the highest level of IFN-γ secretion was observed in the vaccination group with rTα1-BP5 plus H9N2 WIV (P < 0.01) (Figure 5A and B). Taken together, the results suggested that rTα1-BP5 promoted the secretion of both Th1 and Th2 cytokines.
\n
Figure 5.
Effect of rTα1-BP5 to H9N2 AIV vaccination on cytokine production in chicken sera. Chickens were immunized two times, and chicken sera were collected on days 7 and 21 after the first immunization. Cytokine release was measured by using commercial chicken cytokines gamma interferon (IFN-γ) and interleukin 4 (IL-4) ELISA kits. The data presented are means ± SD of results from five replicates. *, P < 0.05, and **, P < 0.01, compared with chickens immunized with H9N2 WIV alone.
\n
Figure 6.
rTα1-BP5 significantly stimulates chicken T- and B-lymphocyte proliferation. Chickens were immunized two times, and chicken thymus and bursa of Fabricius were collected on days 7 and 21 after the first immunization. T- (A) and B (B)-lymphocyte proliferation assays were evaluated by MTT method. The data presented are means ± SD of results from five replicates. *, P < 0.05, and **, P < 0.01, compared with chickens immunized with H9N2 WIV alone.
\n
\n
3.6. rTα1-BP5 significantly enhances T- and B-lymphocyte proliferation
\n
To investigate the effects of rTα1-BP5 on T- and B-lymphocyte proliferation, thymus and BF were collected from chickens immunized with rTα1-BP5 plus H9N2 WIV. T-lymphocyte proliferation responses of chickens immunized with Tα1 plus H9N2 WIV, BP5 plus H9N2 WIV, and H9N2 AIV vaccine were enhanced at 7 days compared with chickens immunized with H9N2 WIV alone (P < 0.05), whereas it was higher for that immunized with rTα1-BP5 plus H9N2 WIV (P < 0.01). On day 21, T-lymphocyte proliferation responses of chickens immunized with Tα1 plus H9N2 WIV and H9N2 AIV vaccine were higher than chickens immunized with H9N2 WIV alone (P < 0.05), while it was highest for that immunized with rTα1-BP5 plus H9N2 WIV (P < 0.01) (Figure 6A). Similarly, B-lymphocyte proliferation responses of chickens immunized with BP5 plus H9N2 WIV and H9N2 AIV vaccine were enhanced at 7 and 21 days compared with chickens immunized with H9N2 WIV alone (P < 0.05), whereas, it was highest for that immunized with rTα1-BP5 plus H9N2 WIV (P < 0.01) (Figure 6B). The data indicated that rTα1-BP5 promoted T- and B-lymphocyte proliferative responses.
\n
\n
3.7. rTα1-BP5 significantly promotes immune protection against H9N2 AIV challenge
\n
To evaluate whether rTα1-BP5 promotes immune protection against H9N2 AIV infection, viral titers in chicken lungs were evaluated at 3, 5, and 7 days after viral challenge by plaque formation assays. Chickens immunized with Tα1 plus H9N2 WIV, BP5 plus H9N2 WIV, and H9N2 AIV vaccine showed significant virus removal from the lungs at 3, 5, and 7 days after challenge compared with H9N2 WIV groups (P < 0.05), while the viral titers of lungs from chicken immunized with rTα1-BP5 plus H9N2 WIV were significantly lower than that in the H9N2 AIV vaccine group (P < 0.01). Moreover, chickens immunized with rTα1-BP5 plus H9N2 WIV had almost no detectable virus particles in the lungs 7 days after challenge (Figure 7A–C). The data indicated that rTα1-BP5 significantly promoted immune protection against H9N2 AIV challenge.
\n
Figure 7.
AIV-viral titers of lung in chickens. Lung samples from individual chicken in each group (n = 5) were collected on days 3, 5, and 7 post challenge with 2.5 × 106 TCID50 avian influenza virus (A/chicken/Jiangsu/JS-1/2002(H9N2)). Each lung sample was diluted to 1 mL with 1640 media. The titers are presented as pfu per mL. The data presented are means ± SD of results from five replicates. *, P < 0.05, and **, P < 0.01, compared with chickens immunized with H9N2 WIV alone.
\n
\n
\n
4. Discussion
\n
In the event of an influenza pandemic, vaccination is one of the most effective ways of intervention in terms of reducing cost, disease, and even death. Appropriate adjuvant can enhance the immunogenicity of the vaccine and improve the immune responses [38, 39]. However, most of the adjuvants used in conjugation with antigen have unacceptable levels of side effects, only a few of them are used clinically [40]. Thus, we need to find new and optimal adjuvant candidates for vaccine. In recent years, some small peptide immunostimulants were reported in use for vaccine adjuvants [41–43]. Both Tα1 and BP5 are associated with immune regulation. Previous studies showed that both Tα1 and BP5 had high potential as an adjuvant for vaccines [26, 44].
\n
In this study, the fusion peptide of rTα1-BP5 was designed and synthesized, and to investigate it as an adjuvant for inducing immune responses in chickens upon vaccination with inactivated H9N2 avian influenza virus (WIV). An effective adjuvant should be able to enhance the levels of both humoral and cell-mediated immunity. To investigate the effect of rTα1-BP5 on humoral responses, chickens were immunized with H9N2 WIV combined with Tα1-BP5, and then titers of HI antibody, antigen-specific antibodies, and AIV-neutralizing antibodies were assessed. Then, we found that rTα1-BP5 significantly enhanced HI antibody and antigen-specific IgG antibodies titers, promoted the secretion of AIV-neutralizing antibodies, which suggested that rTα1-BP5 enhanced the levels of humoral immune responses in chickens when it was co-immunized with H9N2 WIV.
\n
In addition to humoral immune responses, cellular immunity also plays an important role in fighting influenza virus infections [45]. The levels of Th1- and Th2-type cytokines are important references to measure cellular immunity. And lymphocyte homeostasis is required for the maintenance of normal immune function [46]. Th1-type cytokines mainly include IL-2, TNF-α, and IFN-γ, whereas Th2-type cytokines include IL-4, IL-5, and IL-10 [47]. Our study though analyzed the production of Th1 (IFN-γ)- and Th2 (IL-4)-type cytokines, and T- and B-lymphocytes proliferation in vaccinated chickens post immunization to evaluate the cell-mediated immunity. The results suggested that rTα1-BP5 promoted the secretion of both Th1 and Th2 cytokines and T- and B-lymphocyte proliferative responses. Overall, this study found that rTα1-BP5 not only enhanced the humoral immune responses but also promoted the cell-mediated immune responses, and it had the potential to use as an adjuvant.
\n
To further evaluate the influence of rTα1-BP5 as an adjuvant on the immunity protection provided by H9N2 AIV vaccine against AIV infection, chickens were intramuscularly challenged with H9N2 AIV (A/chicken/Jiangsu/JS-1/2002) on day 28 post immunization. After 3 days post challenge, the PBS group chickens that received the challenge virus were mildly depressed. No other clinical signs were observed in that group or any of the other groups, which is typical of low-pathogenicity AIV in chickens [48, 49]. At 7 days post challenge, only the PBS-challenged group had mild, grossly detectable lesions in both the respiratory and gastrointestinal tract. And we found that the viral titers of lungs from chicken immunized with rTα1-BP5 plus H9N2 WIV were significantly lower than all the other groups at 3 days. Chickens immunized with rTα1-BP5 plus H9N2 WIV had almost no detectable virus particles in the lungs at 7 days after challenge. Our data indicated that rTα1-BP5 could effectively inhibit the replication of H9N2 AIV in chickens and promote virus clearance in the lungs of chickens. Thus, rTα1-BP5 had the potential to be used in vaccine formulations to provide improved protection against H9N2 AIV infection in poultry.
\n
In summary, this study demonstrated that inactivated H9N2 AIV vaccine with Tα1-BP5 as an adjuvant enhanced strong immune responses at both humoral and cellular levels against AIV infection in chickens. These data may provide a novel insight to find new adjuvant in vaccines.
\n
\n
Acknowledgments
\n
This work was supported by Grant no. 31101792 from the National Natural Science Foundation of China and no. 2012GGJS-077 from the Foundation for University Key Teacher by Higher Education of Henan Province.
\n
\n',keywords:"thymosin α1 (Tα1), Bursopentin (BP5), fusion peptide, avian influenza vaccine, adjuvant",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/51557.pdf",chapterXML:"https://mts.intechopen.com/source/xml/51557.xml",downloadPdfUrl:"/chapter/pdf-download/51557",previewPdfUrl:"/chapter/pdf-preview/51557",totalDownloads:1429,totalViews:73,totalCrossrefCites:1,totalDimensionsCites:1,totalAltmetricsMentions:0,impactScore:1,impactScorePercentile:59,impactScoreQuartile:3,hasAltmetrics:0,dateSubmitted:"November 23rd 2015",dateReviewed:"May 25th 2016",datePrePublished:null,datePublished:"October 26th 2016",dateFinished:"July 4th 2016",readingETA:"0",abstract:"Thymosin α1 (Tα1) and Bursopentin (BP5) are both immunopotentiators. To explore whether the thymosin α1-Bursopentin (rTα1-BP5) is an adjuvant or not, we cloned the gene of Tα1-BP5 and provided evidence that the gene of Tα1-BP5 in a recombinant prokaryotic expression plasmid was successfully expressed in Escherichia coli BL21. To evaluate the immune adjuvant properties of rTα1-BP5, chickens were immunized with rTα1-BP5 combined with H9N2 avian influenza whole-inactivated virus (WIV). The titers of HI antibody, antigen-specific antibodies, Avian influenza virus (AIV)-neutralizing antibodies, levels of Th1-type cytokines (gamma interferon (IFN-γ)) and Th2-type cytokines (interleukin 4 (IL-4)), and lymphocyte proliferation responses were determined. We found that rTα1-BP5 enhanced HI antibody and antigen-specific immunoglobulin G (IgG) antibodies titers, increased the level of AIV-neutralizing antibodies, induced the secretion of Th1- and Th2-type cytokines, and promoted the proliferation of T and B lymphocyte. Furthermore, virus challenge experiments confirmed that rTα1-BP5 contributed to the inhibition replication of the virus (H9N2 AIV (A/chicken/Jiangsu/NJ07/05) from chicken lungs. Altogether, these findings suggest that rTα1-BP5 is a novel adjuvant suitable for H9N2 avian influenza vaccine.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/51557",risUrl:"/chapter/ris/51557",book:{id:"5294",slug:"steps-forwards-in-diagnosing-and-controlling-influenza"},signatures:"Chen Wang, Chengshui Liao, Wufan Zhang, Deyuan Li and Puyan\nChen",authors:[{id:"182816",title:"Dr.",name:"Chen",middleName:null,surname:"Wang",fullName:"Chen Wang",slug:"chen-wang",email:"wangchen2001@126.com",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:{name:"Henan University of Science and Technology",institutionURL:null,country:{name:"China"}}},{id:"188561",title:"Dr.",name:"Chengshui",middleName:null,surname:"Liao",fullName:"Chengshui Liao",slug:"chengshui-liao",email:"liaochengshui33@163.com",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null},{id:"188562",title:"Mr.",name:"Wufan",middleName:null,surname:"Zhang",fullName:"Wufan Zhang",slug:"wufan-zhang",email:"894027170@qq.com",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null},{id:"188563",title:"Dr.",name:"Deyuan",middleName:null,surname:"Li",fullName:"Deyuan Li",slug:"deyuan-li",email:"lideyuan82@126.com",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:{name:"Nanjing Agricultural University",institutionURL:null,country:{name:"China"}}},{id:"188564",title:"Prof.",name:"Puyan",middleName:null,surname:"Chen",fullName:"Puyan Chen",slug:"puyan-chen",email:"puyanchennj42@126.com",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",institution:null}],sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. Materials and methods",level:"1"},{id:"sec_2_2",title:"2.1. Plasmid, viruses, and reagents",level:"2"},{id:"sec_3_2",title:"2.2. Chicken embryos, animals, and vaccines",level:"2"},{id:"sec_4_2",title:"2.3. Gene cloning and expression of the recombinant fusion peptide Tα1-BP5",level:"2"},{id:"sec_5_2",title:"2.4. Activity testing of fusion peptide Tα1-BP5 (rTα1-BP5) in vitro",level:"2"},{id:"sec_6_2",title:"2.5. Immunization of chickens",level:"2"},{id:"sec_7_2",title:"2.6. Detection of antibodies in serum",level:"2"},{id:"sec_8_2",title:"2.7. Determination of AIV-neutralizing antibodies",level:"2"},{id:"sec_9_2",title:"2.8. Cytokine assays",level:"2"},{id:"sec_10_2",title:"2.9. Lymphocyte proliferation response",level:"2"},{id:"sec_11_2",title:"2.10. Virus challenge experiment",level:"2"},{id:"sec_12_2",title:"2.11. Statistical analysis",level:"2"},{id:"sec_14",title:"3. Results",level:"1"},{id:"sec_14_2",title:"3.1. Expression of the recombinant fusion peptide Tα1-BP5",level:"2"},{id:"sec_15_2",title:"3.2. Activity of rTα1-BP5 in vitro",level:"2"},{id:"sec_16_2",title:"3.3. rTα1-BP5 stimulates significant antigen-specific immune responses",level:"2"},{id:"sec_17_2",title:"3.4. rTα1-BP5 promoted the production of AIV-neutralizing antibody",level:"2"},{id:"sec_18_2",title:"3.5. rTα1-BP5 increases the production of both Th1- and Th2-type cytokines",level:"2"},{id:"sec_19_2",title:"3.6. rTα1-BP5 significantly enhances T- and B-lymphocyte proliferation",level:"2"},{id:"sec_20_2",title:"3.7. rTα1-BP5 significantly promotes immune protection against H9N2 AIV challenge",level:"2"},{id:"sec_22",title:"4. Discussion",level:"1"},{id:"sec_23",title:"Acknowledgments",level:"1"}],chapterReferences:[{id:"B1",body:'Fouchier RA, Munster V, Wallensten A, Bestebroer TM, Herfst S, Smith D, Rimmelzwaan GF, Olsen B, Osterhaus AD. 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DOI: 10.1016/j.vaccine.2006.08.036'},{id:"B46",body:'Wen L, Chen SJ, Zhang W, Ma HW, Zhang SQ, Chen L. hsBAFF regulates proliferation and response in cultured CD4(+) T lymphocytes by upregulation of intracellular free Ca(2+) homeostasis. Cytokine. 2011; 53: 215–222. DOI: 10.1016/j.cyto.2010.11.006'},{id:"B47",body:'Kumar DA, Manikandan P, Sumitra M, Raju KV, Gayathri C, Arutselvan N, Puvanakrishnan R. A novel peptide derivative exhibits anti inflammatory and antioxidant activity in adjuvant induced arthritis in rats. Molecular and Cellular Biochemistry. 2002; 229: 9–17. DOI: 10.1023/A:1017980024029'},{id:"B48",body:'Lee CW, Song CS, Lee YJ, Mo IP, Garcia M, Suarez DL, Kim SJ. Sequence analysis of the hemagglutinin gene of H9N2 Korean avian influenza viruses and assessment of the pathogenic potential of isolate MS96. Avian diseases. 2000; 44: 527–535. DOI: 10.2307/1593091'},{id:"B49",body:'Soda K, Asakura S, Okamatsu M, Sakoda Y, Kida H. 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Key Laboratory of Veterinary Oncological Immunology, Henan University of Science and Technology, Luoyang, China
Key Laboratory of Animal Bacteriology of China’s Department of Agriculture, Nanjing Agricultural University, Nanjing, China
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1. Introduction
Perovskites, which have the same crystal structure as calcium titanium oxide (CaTiO3), are semiconductor materials that have gained massive interest in various technology. When exposed to light, the structure and characteristics of these materials allow them to transfer electric change. These materials are very beneficial for system memory and memory storage in computer memory. Perovskites’ resistive switching (RS) properties enable fast writing speed and long durability. Thus, RS is employed in the most recent computer memory technology, Resistive Random Access Memory (ReRAM), which is expected to replace flash memory [1, 2].
In memory devices, there are many forms of RS. Bipolar and unipolar switching, write-once-read-many (WORM) [3], and multilevel RS [4] are examples of these. This chapter will discuss the advancements in the use of perovskites for RS memory. Oxide perovskites, halide perovskites, and layered perovskites are some of the perovskite materials employed in this application. Lastly, some design challenges are discussed, and future work is proposed.
2. System memory & memory storage technology
Digital technology is being employed extensively in today’s economy, and it shows no signs of abating. As a result, the progress of system memory and memory storage has accelerated, resulting in new improvements. The system memory is where the computer stores currently running applications and data. On the other hand, memory storage is generally for the goal of orderly retrieval and documentation. Figure 1 depicts the chronology of the evolution of system memory and memory stage.
Figure 1.
A timeline depicting the progress of system memory and memory storage over time.
In 1932, Gustav Tauchek invented the drum memory technology which marked the beginning of system memory [5, 6]. The drum memory was cylindrical in form, with an outside covering comprised of recordable ferromagnetic elements, and it could store up to 500,000 bits, or 62.5 kilobytes of memory [7]. Eventually, in the mid-1940s, the delay line memory was found, which was a refreshable memory that used sequential access and was constructed of mercury. This unique mercury delay line was capable of transmitting data at a rate of around 5,000,000 binary digits per second. It was not until World War II that the United States Navy adopted the initial drum memory idea and refined it into the magnetic drum memory system. The magnetic core memory was then constructed using tiny toroidal ferrimagnetic ceramic ferrites. The memory was stored via an induced magnetic field, which could store one bit depending on the magnetization direction [8]. Twister memory, which used magnetic tape instead of rings to replace core memory, was introduced in 1968 at Bells Lab but received little attention [9]. The magnetic tape was intentionally chosen to enable magnetization only down the length of the tape. As a result, only one point of the twistor would have the proper field direction to ever get magnetized. On the other hand, bubble memory which is a sort of non-volatile memory employs a small layer of magnetic material in its fabrication due to the influence of an external magnetic field. This contains little magnetized patches known as bubbles or domains, each of which may retain one bit of data [10, 11]. Similarly, bubble memory also suffered the same fate as twister memory since both were eclipsed by the development of dynamic RAM.
The invention of dynamic Random-Access Memory (RAM) in the 1960s laid the groundwork for today’s multibillion-dollar memory technology sector. Every sort of memory technology described above is rendered obsolete by the discovery of RAM. The earliest architecture of dynamic RAM was a square array with a capacitor and a transistor for each data bit [12]. In today’s technology, a broad range of RAM technologies have been researched for their commercialization potential. The advancement of memory storage technology began in 1976 with the usage of punched cards, with certain holes on them as a set of instructions for digital programs [13]. It was then refined further into punch tapes or paper tapes. Similarly, paper tapes were developed to replace punched cards, which were considerably more convenient since they provided a continuous set of data or instructions without the need to insert punched cards one at a time. The substance used to create the paper tapes was then altered and replaced with magnetic materials.
Magnetic tapes were significantly easier to use and could contain far more data than paper tapes. This has transformed the broadcasting industry by allowing live broadcasts to be recorded and replayed at any time [5]. It was not until 1969 that memory storage technology was substantially influenced by the invention of magnetic discs, which can store databases and vast volumes of data. As a result, floppy discs were inspired by magnetic discs, which were portable and generally available to the public. Flash drives, which are based on Erasable Programmable Read Only Memory (EPROM) and Electrically Erasable Programmable Read Only Memory (EEPROM) technologies, are no longer rare in today’s technology [14, 15]. Future predictions for system memory and memory storage technologies have focused on a few advancements, including the ReRAM technology, which was projected to replace flash memory.
3. Resistive switching in memory devices
3.1 Bipolar & unipolar switching
The rapid switching speed, lower power consumption, and excellent scalability, ReRAM has emerged as the most encouraging choice for the future use of non-volatile devices [1, 2]. A dual terminal ReRAM device has an insulating layer wedged between two conducting electrodes. An external electric field can cause the memory cell to flip between two resistance states, known as the low resistance state (LRS, ON state) and high resistance (HRS, OFF state) state [16]. ReRAM is divided into two switching modes: unipolar and bipolar. The polarity of the switching voltage is inconsequential in unipolar switching, whereas in bipolar switching, the electrical polarity required to change from an HRS to an LRS is the inverse of that required to switch from an LRS to an HRS. Switching materials such as organics, binary oxides, and perovskite oxides, have been studied to achieve ReRAM with reliable switching, high ON/OFF current ratio, and long retention period [17, 18, 19].
Non-volatile memory (NVM) is a classification of computer memory that cannot be deleted or removed even when the power is turned off. In today’s technology, NVMs are typically utilized for long-term data storage or secondary storage. Flash drives, magnetic storage devices, ferroelectric RAM, magnetoresistive RAM, ReRAM, and optical discs are examples of NVM devices. In today’s storage technology, NAND (Boolean operator and logic gate) flash memory are widely used in most goods. However, the demand for faster writing speeds, higher density, and lower cost drives new research into developing technologies such as ReRAM. In the late 1990s and early 2000s, researchers investigated ReRAM technology, which allows RS between two resistance states to be exchanged via a thin film layer [20]. It works on the principle of applying a high voltage across a dielectric to transition from insulating to conductive qualities via a conduction filament pathway. Classification of ReRAM mechanism as memristors is controversial [21]. Theoretically, their RS curves differ from each other. Figure 2 shows a typical current-voltage (I-V) characteristic obtained for a ReRAM device [2]. Such classifications of ReRAM and memristors are still a source of contention today.
Figure 2.
(a) The predicted and typical types of (a) unipolar switching and (b) bipolar switching ReRAM curves. Adapted from Figure 4 [2].
The memristor is the fourth fundamental circuit element identified, after resistors, capacitors, and inductors. Chua et al. suggested the notion of memristor in 1971 [22], and it was validated in 2008. There are several materials having memristive characteristics which have been discovered since 1962. Memristors, like resistance in Ohms, are defined as:
M=dφdqE1
where φ is magnetic flux and q is electric charge. As observed in its theoretical memristor curves, memristor curves are typically non-linear devices. Real-world memristors, on the other hand, have a comparable I-V characteristic to ReRAM. Some researchers hypothesized that such behavior was caused by conducting filament [23], active memristor [24], and non-zero crossing [25]. ReRAM curves, on the other hand, can be classified as bipolar or unipolar switching. To generate the RS curve, unipolar switching uses the same polarity of the swept bias with variable magnitudes, but bipolar switching requires various polarities [26]. ReRAM devices, in general, consist of an insulator layer sandwiched between two electrodes. Conduction pathways were formed by the flow of charge carriers alternating between the cathode and anode, which was induced by several physical mechanisms that are still commonly utilized today. Furthermore, the conduction methods differ depending on the materials utilized and the device’s overall architecture. Szot et al. were the first to detect conductive filament using an electron microscope, which they attributed to filament build-up and rupture [27].
3.2 Write-once-read-many (WORM)
WORM devices, in general, are devices in which data that has been written cannot be manipulated or removed. WORM memory is used in fields such as healthcare, security, taxation, and accounting where the data cannot be tampered with or updated to secure information. The most prevalent WORM devices in use today are the Compact Disc Recordable (CD-R) and Digital Versatile Disc Recordable (DVD-R). Furthermore, the “read-many” element implies that the device’s data can be read an infinite number of times, with the only limitation being the device lifetime. The WORM memory pixels are read according to the rows, with an unwritten pixel labeled as logical “0” and a written pixel labeled as logical “1” [3].
Today’s WORM memory technology is based on the electrically or laser programmed fuse WORM type. However, in current WORM research, the emphasis was shifted to organic materials or solution process techniques in order to achieve rapid switching rate, lower power consumption,, large storage density, simplicity, and being cost-effective, which has been dubbed WORM RS [28, 29]. Unlike ReRAM, the intrinsic features of WORM RS were sufficient to oppose the applied electric field, resulting in an irreversible shift. A typical WORM RS I-V curve obtained when a voltage is supplied is shown in Figure 3 [30]. The change between resistance states (OFF to ON) is an irreversible process, indicating WORM features.
Figure 3.
A typical WORM curve transiting from OFF to ON process. Adapted from Figure 3 [30].
3.3 Multilevel resistive switching
The potential of multilayer RS effect has been documented in several inorganic materials due to its superior memory performance, but the difficult fabrication method and stiffness restrict the development for ReRAM [31, 32, 33]. Organometal halide perovskites (OHPs) have recently sparked a lot of interest in the ReRAM community because to the high flexibility, variable band gaps, huge absorption coefficients, and long electron-hole diffusion length [34, 35]. Furthermore, OHPs highly feature defect-tolerant, simple, and cost-effective solution-processed procedures for fabricating the OHPs layers. As a result, they envision it being used in multilevel RS, which is advantageous for multilevel data storage. Typically, these devices feature numerous resistance states that can be changed within the device. Figure 4 shows a typical multilevel perovskite memory consisting of a all-inorganic CsPb1–xBixI3 perovskite film [36]. This reveal that multilevel RS was accomplished by altering the reset stop voltages.
Figure 4.
I-V curves of Au/KCl-MAPIC/ITO/glass indicating two VSETs of 0.8 V and 1.0 V. Adapted from figure 3 [36].
4. Recent advancements of perovskites in memory devices
4.1 Oxide perovskites in memory devices
In recent oxide perovskites based works, a Ag/BaTiO3/Nb:SrTiO3 ferroelectric tunnel junction (FTJ) with the quickest switching speed of 600 ps has been constructed [37]. When the sub-nanosecond switching action is maintained at 112.85°C, the device exhibits great temperature resilience. In addition, the gadget established 32 states or 5 bits of states for each cell, which is regarded to be the highest in the class. The combination of a high carrier concentration in the Nb:SrTiO3 electrode and the low work function of silver metal has resulted in significant increases in operation speed with a low current density of 4 × 103 A cm−2. The device performance can be seen through the rapid resistance switching at normal temperature depicted in Figure 5.
Figure 5.
(a) Graph of resistance vs. driven voltage (Vd), and (b) common resistance switching between ON state and various high-resistance states at pulse duration, td = 600 ps. Adapted from Figure 2 [37] .
The advancement of fabrication for BaTiO3 has been established by growing this material on pre-deposited SrTiO3 substrate using the epitaxy technique to form a free-standing film. Then, they are transferred onto a silicon substrate for integration into complementary metal-oxide-semiconductor (CMOS) devices [38]. Not only the performance of the device is comparable with previously reported work [39], but it also exhibits a large ON state current with good bias voltage measurement of 0.2 V. This enables a non-destructive readout during the operation process. With an optimal depiction of SrRuO3/BaTiO3 dual buffer layers, highly structured BiFeO3 films were prepared oriented on flexible mica substrates [40]. The BiFeO3 films possess a stable ferroelectric polarization with over 100 bending cycles at a 5 mm radius. Therefore, it results in the continuously controllable resistance memristor characteristics which suggests the feasibility for solid synaptic devices. Moreover, using interactive supervised learning, the handwritten digits reveals excellent recognition accuracy valued at 90% in artificial neural network simulations which displays the potential for flexible ferroelectric memristors in wearable devices (data storage and computation). Ferroionic tunnel junctions have been suggested to make a huge electroresistance in ReRAM based BaTiO3 [41]. In low-resistance states, it works as a ferroelectric tunnel junction and as a Schottky junction which is due to changes within the interface caused by a field. This device significantly employs the ferroelectric barrier (BaTiO3) and Nb-doped SrTiO3 as the bottom electrode. The giant electroresistance result in ON/OFF ratios of 5.1 × 107 and 2.1 × 109 at room temperature and 10 K, respectively. The movement of oxygen vacancies from polarization reversal caused by the bias voltage could significantly alter the dimension of the interface barriers [42].
The ferroelectric manipulation of spin-filtering BaTiO3/CoFe2O4 composite barriers has been demonstrated in multiferroic FTJ synapses [43]. By manipulating the polarization switching of BaTiO3, it is possible to establish long-term memory and a constant conductance change achieving a 544,400% ON/OFF current ratio. On a crossbar neural network, supervised learning simulations applies the Spike-Timing Dependent Plasticity (STDP) outcomes as a database for weight training which achieved recognition accuracy rates above 97%. As a result, there is an approximately 10-fold shift in tunneling magnetoresistance ratio including a turnaround relying on the resistance state of the electrodes when the polarization is switched. A novel approach to multiferroic neuromorphic devices with energy-saving electrical exploitation is provided by these studies, notably the switchable spin polarization. Additional FTJ device options include ferroelectric oxide-grown spinel ferrite barriers, which open a wider range of potential applications.
On the other hand, SrTiO3 has been used to develop the Pt/CeO2/Nb: SrTiO3 heterostructure which demonstrates outstanding memory behavior with a highest RS ratio of 3 × 104 [44]. Under the irradiation of an ultraviolet 405 nm laser beam, an obvious photoresponse was detected, which also corresponds to substantial switching characteristics in a high resistance state. This device demonstrates light-manipulated RS and voltage dependent photoresponse, which are two different types of RS. The RS and photoresponse features were contributed from the Schottky barrier at the Pt/CeO2 interface, as well as the electron trapping, and de-trapping caused by oxygen vacancies at the interface. An ultrathin (6.2 nm) ferroelectric La0.1Bi0.9FeO3(LBFO) layer has been introduced on a 0.7 wt% Nb-doped SrTiO3 (001) single-crystal substrate to form a Pt/La0.1Bi0.9FeO3/Nb-doped SrTiO3 heterostructure [45]. The ferroelectricity of the LBFO film was extremely high, but the coercive field was extremely low. By adjusting the thickness of the LBFO film, it was possible to produce a high resistance OFF/ON ratio of up to 2.8 × 105 for the Pt/LBFO (6.2 nm)/NSTO heterostructure. Moreover, the heterostructure exhibited multi-level storage and outstanding retention properties, as well as steady bipolar resistance switching behavior, which is suitable for application in ferroelectric memristors. On the LaBiFeO3/Nb-doped SrTiO3 interface, the resistance switching behavior has been demonstrated to be caused by a modulating impact of ferroelectric polarization turnaround on both the breadth of the depletion area and the potential barrier’s heigh.
Using pulsed-laser deposition (PLD) technology, epitaxial BiFeO3 (BFO) thin films were fabricated to produce the robust in-plane domain dynamic process created when applied under the influence of external electric fields [46]. It has also been noticed that the retention and repeatability are good, particularly at high temperatures. Besides, by forming a heterostructure consisting of indium tin oxide (ITO), BFO, and strontium ruthenium oxide (SRO), an optically triggered non-volatile memory has been demonstrated [47]. In comparison to traditional devices, in which optical excitations often increase conductivity, the constructed structure demonstrates a significant drop in conductivity (1 × 10−4) following laser illumination at wavelengths 405, 532, and 1064 nm, indicating that the device is poorly conductive. Additionally, optical stimuli may be used to reset the negative optoelectronic memory, and an electrical pulse could be used to establish the memory. It was discovered that this property could be inhibited by annealing in an oxygen-rich environment, but that it could be restored by annealing in an oxygen-depleted atmosphere. Based on investigations of the transport and dielectric characteristics, it has been determined that the optical/electrical RS behavior observed at the ITO/BFO interface is caused by the potential profile’s modulation at the ITO/BFO interface caused by optical and electrical excitations.
Fundamental research into the reversible topotactic phase change between the insulating brownmillerite (BM) phase and the conducting perovskite structure is critical for the creation of RS memories. Using SrFeOx as a model, the system demonstrated that in the ON state, SrFeO3 nanofilaments are produced and stretch essentially through the BM SrFeO2.5 matrix, and that in the OFF state, they are ruptured, indicating indisputably the presence of a filamentary RS process [48]. The nanofilaments are roughly 10 nm in diameter, permitting for the first time the downscaling of Au/SrFeOx/SrRuO3 RS devices to the 100 nm range. They have exceptional performance, with an ON/OFF ratio of up to 104, a retention time of more than 105 s, and an endurance of up to 107 cycles, among other things.
On the other hand, an investigation into the process of irreversible RS conversion from bipolar to unipolar process is conducted in a capacitor model composed of SrZrO3, TiOx/Pt, which has been produced on a substrate composed of Pt, Ti, SiO2, and SiO2 [49]. The I-V properties of bipolar RS memory were seen in the RS operating voltage range spanning from +2.5 V to −1.9 V, and the memory exhibits outstanding durability and retention characteristics. An additional forming step happens when the voltage bias is raised above +4 V, irrevocably changing the RS mode from bipolar to unipolar. This process occurs as the voltage bias is raised above +4 V. In this study, two materials are combined with two different switching processes to create an RS memory with acceptable properties in various current regions. Furthermore, a bipolar RS characteristic was investigated in a Pt/LaNiO3/Nb: SrZrO3/Cu structure, where the Cu and LNO layers function as capping and buffering layers, respectively [50]. It was feasible to achieve a high endurance performance for the bistable bipolar switching characteristic at room temperature, which was measured up to 1.2103 times. The Cu layer was utilized as a reservoir layer to change the distribution of oxygen vacancies and traps inside the films, resulting in a steady RS response, low operation voltage, and extended retention duration.
A nanocomposite consisting of La0.7Sr0.3MnO3 (LSMO) and reduced graphene oxide (rGO) has been identified as a viable option for non-volatile memory applications in oxide electronic devices [51]. Individual component phases were identified because of the structural characterization process. A minimal switching speed of 1.1 μs and a relatively steady switch mechanism over 1000 switching cycles were obtained from the device. The switching behavior is also shown to be resilient against variable voltage sweeping rates. It is highlighted that the transportation of the oxygen ions at the SET and RESET voltages result in alterations of the resistance states due to the conduction filaments forming or rupturing. LSMO is also combined with BTO to form a heterostructure of non-volatile and reversible RS [52]. It was revealed that altering the electric field orientation caused variations in the LSMO layer’s resistivity and metal-insulator transition temperature (TMI). When the BTO layer is subjected to a negative electric field, the resistivity for the accumulation state of hole carriers drops while the TMI for the accumulation state of hole carriers rises. When a positive electric field is applied to the BTO layer, the resistivity rises while the TMI falls for the hole carrier depletion condition.
4.2 Halide perovskites in memory devices
An organo-metal halide source has been shown to produce perovskite layers through one-step spin-coating technique for the construction of unipolar RS devices in a cross-bar array design utilizing a simple one-step spin-coating procedure [53]. With gold as the electrodes, these unipolar perovskite RS devices attain a high ON/OFF ratio of up to 108 while operating at a small operating voltage, with high stability, over 1000 writing cycles, and retention over 104 s. The memory devices were successfully incorporated into an 8 8 crossbar array design with up to 94 percent yield. Furthermore, as shown in Figure 6, the 1D-1R system of selective activation of memory cells was shown by eliminating crosstalk interference between nearby cells linked by external diodes.
Figure 6.
A diagram of the identical reading procedure in a 2 × 2 array in a 1D-1R architecture with memory cells connected by external diodes. Adapted from Figure 4 [53].
An active layer for resistive memory has been developed using CsPbBr3 single-crystal film (SCF) [54]. With an extremely high switching ratio of above 109 and a rapid switching speed of 1.8 s, the Ag/CsPbBr3/Ag memory cells demonstrate repeatable RS. A large interface contact is produced when the metal/CsPbBr3 SCF contact has an interface S value of 0.50, indicating that a large interface contact has been established. Because of the high RS ratio at the interface, the high interface contact leads to the stable high resistance state (HRS), and the steady HRS leads to an ultrahigh RS ratio. Besides, it has been demonstrated that the use of vacancy defects in lead halide perovskite structures may create excellent performance nano floating gate memory (NFGMs) [55]. A CdS nanoribbon (NR) surface was evenly covered with CH3NH3PbBr3 nanocrystals (NCs) using a simple dip-coating procedure, resulting in a core-shell structure composed of CdS NR/CH3NH3PbBr3 NC cores and shells. It is noteworthy that the device exhibited a very large memory window of up to 77.4 V and a long retention time of 12,000 s, as well as a high current ON/OFF ratio of 7 × 107 and long-term air stability for 50 days, all of which were attributed to the presence of sufficient carrier trapping states in CH3NH3PbBr3 NCs.
On the other hand, a CH3NH3PbI3−xClx/FTO RS device structure has been proven to retain information in dual levels of resistance states generated by electrical probe stimulation [56]. The device with the silver probe demonstrates bipolar RS behavior after formation, with a 106 ON/OFF resistance ratio, showing that it is bipolar RS. The constructed probe-based memory cell has a minimum endurance of 104 cycles and a minimal retention length of 2 × 103 s, which are both good performance features. Thus, organic-inorganic lead halide perovskite (OILHP) materials are highly proposed as a feasible candidate for usage as a storage layer for probe-based storage memories. Thin polyethyleneimine (PEI) interfacial layers have been introduced between the layers to avoid direct contact between the perovskite layer and the top and bottom electrodes, resulting in a device structure consisting of ITO/PEI/CH3NH3PbI3/PEI/metal [57]. This device can reach over 4000 durability cycles while maintaining a low operating voltage of around 0.25 V. Aside from that, the repeatability of memory switching behavior was proven across 180 devices manufactured using eight different device batching settings.
Recently, 2D/3D perovskite heterostructure films consisting of 2D perovskite (phenethylammonium lead iodide, PEA2PbI4)/3D perovskite (MAPbI3) have been produced using a low-temperature all-solution technique [58]. The integration of 2D and 3D perovskite RS memories displayed remarkable efficiency, with an overall durability of 2700 cycles, an ON/OFF ratio of more than 106, and a reaction time of 640 μs. Both the expected activation energy for thermally aided ion hopping and the time-of-flight secondary ion mass spectrometry data suggested that the 2D perovskite layer effectively blocked Ag ion migration through into the 3D perovskite film. By putting n-butylammonium iodide above CH3NH3PbI3−xClx (MAPbI3−xClx), another 2D/3D memory device was demonstrated [59]. The perovskite film is made in a single step by heating molten salt methylammonium acetate to room temperature and spinning it in the air. When compared to their 3D counterparts, RS memory devices with a 2D/3D perovskite heterostructure provide a significantly better switching window with an ON/OFF ratio of more than 103 while needing a lower operating voltage. The 2D/3D perovskite heterostructure is advantageous for manufacturing uniform-crystalline-grain, highly compact structures, and it can passivate defect states for the MAPbI3−xClx film and interface, resulting in improved memory properties for both the film and the interface.
In contrast, the cube of CsPbX3 was used in an Al/CsPbClxBrx (x = 3, 1.5, 0)/ITO/PET memory device that demonstrated a bipolar RS pattern at a low working voltage [60]. When compared to all other memory devices developed, the CsPbBr3-based system has the most obvious RS qualities, such as the lack of an initial forming procedure, reproducibility, uniform switching, and a long retention period with a high ON/OFF ratio. The multilayer data storage potential of flexible memory devices may also be evaluated by making minor changes to current compliance and stopping voltage.
Recently, flexible wearable electronic materials and fiber-shaped resistive random access memory based on MAPbI3, an organic-inorganic halide perovskite semiconductor, have been created utilizing a simple and cost-effective cheap deep coating process [61]. After refining the manufacturing settings, a well-arranged pinhole-free layer was coated on the aluminum fiber. The device has a bipolar RS feature with a roughly 106 ON/OFF ratio, a low working voltage, and a retention duration of more than 104 s. More crucially, the switching mechanism has remained nearly unchanged with up to a 45° bending angle.
In addition, our previous study investigated increasing the molar ratio of Pb to Ti by 5% in the MAPbI3-TiO2 layer, which increased VSET and VRESET values to 3.6 V and 1.1 V, respectively [59]. This study is regarded as a forerunner in understanding the use of a single layer MAPbI3-TiO2 in several applications. In the meanwhile, Cs3Sb2I9 inorganic halide perovskite has been developed as a lead-free source of high ReRAM and artificial synaptic devices [62]. A vapor-assisted solution technique was used to create this 2D perovskite (VASP). The memristive devices not only feature reproducible bipolar RS with a massive ON/OFF ratio of 104 at a low working voltage of 0.4 V, but they also have superb retention over 104 s and extraordinary resistance to environmental degradation. The ReRAM devices show promise to produce phototunable memories and artificial synaptic devices with the capacity to perform concurrent processing and learning due to high light-matter interaction in the perovskite and an intrinsic electronic–ionic connection.
4.3 Layered perovskites in memory devices
Dion-Jacobson organic-inorganic halide perovskite (OIHP) has been proven as a resistive switching memory (RSM), with grain size varying to improve grain boundaries [63]. By adjusting the ratio of N, N-dimethylformamide to dimethyl sulfoxide in the reaction mixture, the grain structure of the OIHP may be easily controlled. The controlled grain sizes in RSM can alter the paths for halide ion migration, enabling for a shift in the ON/OFF ratio by modifying the grain size. Large memory applications also need the use of the cross-point array structure. However, because sneak-current paths may generate undesirable current flow across unselected memory cells in a cross-point array structure, it is critical to minimize leakage current from surrounding cells by integrating selector devices in the design. We demonstrate the usage of selector devices in combination with the devices to avoid sneak current paths in OIHP-based RSMs. These findings suggest that OIHP might be utilized in high-density memory applications.
An Al2O3/2D Ruddlesden-Popper perovskite (2D PVK) heterostructure dielectric architecture, on the other hand, has been utilized to create ambipolar SnO transistor-based non-volatile memories with multibit memory behavior and ultralong retention time of >105 s [64]. The unique storage features are attributed to the decreased gate leakage generated by the Al2O3 layer, as well as the hopping-like ionic transport in 2D PVK with changeable activation energy under various light intensities. Because of the photoinduced field-effect process, it is feasible to operate a top-gated transistor in the presence of light, which would not be possible in the absence of light. As a result, it has exceptional photoresponsive properties, such as an extremely high specific detector detectivity of 2.7 × 1015 Jones and a large bandwidth spectrum differentiating capacity (375–1064 nm). The shape of metal halide perovskite layers, rather than grain size, is critical for high-performance memory systems. By including the organic semiconductor 2,7-dioctyl[1]benzothieno[3,2-b]benzothiophene (C8-BTBT) into the perovskite formulation, the microstructure of solution-processed layered Ruddlesden–Popper-phase perovskite films based on phenethylammonium lead bromide ((PEA)2PbBr4) can be manipulated [65]. The hole is transported in the CB-BTBT, while the charge is stored in the (PEA)2PbBr4. With the combination of the (PEA)2PbBr4/C8-BTBT channels, the transistor-based memory device exhibited a huge record memory window over 180 V, large erase/write channel current ratio 104, excellent data retention, and hood durability over 104 cycles. Moreover, a Ruddlesden-Popper-phase strontium titanate, SrO(SrTiO3)n (n = 1) and conventional perovskite SrTiO3 have been combined to form a heterojunction thin-film on an FTO substrate using the sol-gel method [66]. The Au/Sr2TiO4/SrTiO3/FTO/glass memory device performed a stable switching ratio over 102 under a high operating voltage of 8 V.
Generally, ion migration in the I-V hysteresis is known as the drawback of halide perovskite optoelectronic. This unwanted issue has been solved by employing layered Ruddlesden-Popper perovskites (RPPs) [67]. As a result, the memory devices of RPP with indices n = 5 show the largest ON/OFF ratio of 104, operated in low VSET, in comparison to n = 1 and the 3D indices composition of perovskite, as presented in Figure 7 In addition, the device can last for 500 cycles in an inert environment with data retention of 250 h. The data retention can be extended when the device is operated under 60% relative humidity. These results are due to the chemical interaction between moving ions and the external contacts, which results in a modification of the charge transfer barrier at the interface, which subsequently modifies the device’s resistive states.
Figure 7.
Electrical characteristics of various ReRAM devices: (a) I-V curves for devices incorporating perovskites with varying n indices (n = 1, 5/3D) in an ITO/PEDOT:PSS/perovskite/PCBM/Ag setup (b) overview of SET voltages and ON/OFF ratios for various devices. Adapted from Figure 3 [67].
On the other hand, neuromorphic computing requires extremely minimal operating energy to provide huge parallel data processing that mimics the human brain. It is necessary to attain this aim by using resistive memory that is based on materials that have good ionic transport and operate at very low currents. Extremely low operating current facilitates low-power operation by minimizing program, erasing, and read currents. The mixed electronic and ionic transport, as well as the ease with which they may be produced, make the 2D Ruddlesden-Popper phase hybrid lead bromide perovskite single crystals appealing materials for low operating current nanodevice applications [68]. The migration of bromide ions across the exfoliated 2D perovskite layer demonstrates ionic transport in the layer. Resistance memories with the lowest program currents down to 10 pA with a 100 ON/OFF ratio. Furthermore, the resistive memory demonstrated 400 fJ/spike synaptic functioning, which is comparable to the energy consumption required to convey information in the normal nervous system.
The use of lead-free perovskite through Aurivillius phase thin films have been suggested to improve their ferroelectric characteristics by modifying the growth, texture, and orientation [69]. In particular, liquid injection chemical vapor deposition (LI-CVD) was used to grow c-plane oriented Bi6Ti3Fe2O18 (B6TFO) functional oxide Aurivillius phase thin films on c-plane sapphire substrates, which were then annealed at 850°C to generate highly crystalline, well-textured single-phase Aurivillius plate-like shapes with 110 nm average film thickness and 24 nm roughness. Piezoresponse force microscopy (PFM) shows in-plane polarization enhanced by adjusting the deposition of a-axis oriented grains along the plane of the B6TFO films. Interestingly, the device shows a large and stable ferroelectric polarization switching under high operating temperatures of up to 200°C even after 20 h of PFM scanning. These investigations show the promise of B6TFO thin films for high-temperature piezoelectric applications and non-volatile ferroelectric memory applications.
It is also important to highlight that multiferroic materials with associated ferroelectric and ferromagnetic order characteristics might be used to store data by writing bits electrically and reading them magnetically. For example, Aurivillius phase Bi6Ti2.8Fe1.52Mn0.68O18 (B6TFMO) produced by chemical solution deposition (CSD) shows magnetoelectric coupling at ambient temperature [70]. The in-plane ferromagnetic signature can be enhanced by manipulating the deposition method using the liquid injection chemical vapor deposition technique, which is related to the formation of Aurivillius phase [71]. Under magnetoelectric coupling, the ferroelectric switching volume increased by up to 14 percent as compared to CDS-grown films, and irreversible and reversible magnetoelectric domain switching was observed. This demonstrates that B6TFMO thin films are a viable choice for in-plane RAM applications as well as future high data storage multistate memory devices.
5. Design challenges of perovskites based memory devices
Understanding the RS properties is critical when building a RS device. According to published research, RS is often associated with oxygen or halide vacancies, metallic defects, and dislocations in perovskite thin films [72]. The kind of perovskites, film thickness, inclusion of dopants, and selection of bottom/top electrodes are all basic elements to consider in memory design construction. To fabricate a stable and efficient perovskite film device, preparation processes of perovskite thin films and understanding of the interaction between each layer are critical.
Wearable gadgets are gaining popularity among researchers in the age of wearable technology. Conventional electrodes, such as metals or transparent conducting oxides, are inflexible and readily shattered under stress. On the other hand, perovskites may be prepared via low temperature solution processing methods, Flexible devices could be considered while memory performance is maintained as published for study involving polyethylene naphthalate (PEN) or polyethylene terephthalate (PET) substrates [73, 74].
For further development of halide perovskites with high performance RS memory, the stability issue must be addressed due to the sensitivity to heat and moisture. This obstacle must be overcome to compete with metal oxide-based memory, which are more stable and easier to manufacture. To slow down the deterioration process while boosting stability, thick encapsulation might be applied to the devices. This layer may consist of a thin metal oxide or polymer-based layer [75]. Similar, to perovskite solar cells, the usage of lead compound is a key drawback in halide perovskite-based RS devices. Since lead is a poisonous chemical, the environmental impact of lead leaking may be disastrous. As a result, researchers are investigating lead alternatives such as lead-free perovskites. One example that researchers are looking at is tin-based perovskites, which have similar RS capability. Ji et al. discovered a lead-free all-inorganic cesium tin iodide perovskite (CsSnI3) [76]. The (Ag or Au)/PMMA/CsSnI3/Pt/SiO2/Si bipolar RS could represent an ecologically acceptable option.
6. Conclusions
RS memory is one of the most sophisticated techniques for next-generation storage class memory, with lower power consumption, high density, and better performance. RS devices are regarded as one of the viable technologies for next-generation non-volatile memory. In addition to the well-studied usage of perovskite in perovskite solar cells, the use of perovskites in memory devices might be a fascinating subject to examine. Perovskites of various classes, such as perovskite oxides, perovskite halides, and layered perovskites, can be used in various RS devices. There are still numerous options to investigate for RS devices. The interactions of these perovskites with other elements are currently understudied and need to be investigated further. However, they still face significant challenges in entering the commercial sector. Although memory performance is advancing at a rapid pace, fundamental challenges in stability, reproducibility, and real-world applications are being addressed. As a result, perovskite may continue to play a significant role in dominating the memory storage sector soon.
Acknowledgments
This work was financially supported by the AUA-UAEU Joint Research Grant Project (IF016-2021 and G00003485), UNITEN BOLD grant J5150050002/20021170, Fundamental Research Grant Scheme (Project No.: FP113-2019A), Geran Putra-Inisiatif Putra Muda (GP-IPM/2018/9667000) and SATU Joint Research Scheme (ST002-2021).
\n',keywords:"resistive switching, memory devices, memory storage, perovskites, non-volatile",chapterPDFUrl:"https://cdn.intechopen.com/pdfs/82141.pdf",chapterXML:"https://mts.intechopen.com/source/xml/82141.xml",downloadPdfUrl:"/chapter/pdf-download/82141",previewPdfUrl:"/chapter/pdf-preview/82141",totalDownloads:20,totalViews:0,totalCrossrefCites:0,dateSubmitted:"April 22nd 2022",dateReviewed:"May 10th 2022",datePrePublished:"June 7th 2022",datePublished:null,dateFinished:"June 7th 2022",readingETA:"0",abstract:"Although perovskites are widely employed in other industries such as photovoltaics and light-emitting diodes (LEDs), digital technology is rapidly gaining pace in today’s market and shows no signs of abating. As a result, the progress of system memory and memory storage has accelerated into new inventions. The invention of dynamic Random-Access Memory (RAM) in the 1960s laid the groundwork for today’s multibillion-dollar memory technology sector. Resistive switching (RS) capabilities of perovskite-based materials such as perovskite oxides and metal halides have been extensively studied. Chemical stability, high endurance, quick writing speed, and strong electronic interaction correlation are some of the benefits of employing perovskites in RS devices. This chapter will investigate the progress of system memory and memory storage employing perovskites, the advantageous properties of perovskites utilized in memory devices, the various types of RS employing perovskites, as well as the research challenges that perovskite-based memory systems face in future commercial development.",reviewType:"peer-reviewed",bibtexUrl:"/chapter/bibtex/82141",risUrl:"/chapter/ris/82141",signatures:"Gregory Thien Soon How, Mohd Arif Mohd Sarjidan, Boon Tong Goh, Boon Kar Yap and Eyas Mahmoud",book:{id:"11469",type:"book",title:"Recent Advances in Perovskite Materials",subtitle:null,fullTitle:"Recent Advances in Perovskite Materials",slug:null,publishedDate:null,bookSignature:"Dr. Poorva Sharma and Associate Prof. Ashwini Kumar",coverURL:"https://cdn.intechopen.com/books/images_new/11469.jpg",licenceType:"CC BY 3.0",editedByType:null,isbn:"978-1-80355-319-1",printIsbn:"978-1-80355-318-4",pdfIsbn:"978-1-80355-320-7",isAvailableForWebshopOrdering:!0,editors:[{id:"318155",title:"Dr.",name:"Poorva",middleName:null,surname:"Sharma",slug:"poorva-sharma",fullName:"Poorva Sharma"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}},authors:null,sections:[{id:"sec_1",title:"1. Introduction",level:"1"},{id:"sec_2",title:"2. System memory & memory storage technology",level:"1"},{id:"sec_3",title:"3. Resistive switching in memory devices",level:"1"},{id:"sec_3_2",title:"3.1 Bipolar & unipolar switching",level:"2"},{id:"sec_4_2",title:"3.2 Write-once-read-many (WORM)",level:"2"},{id:"sec_5_2",title:"3.3 Multilevel resistive switching",level:"2"},{id:"sec_7",title:"4. Recent advancements of perovskites in memory devices",level:"1"},{id:"sec_7_2",title:"4.1 Oxide perovskites in memory devices",level:"2"},{id:"sec_8_2",title:"4.2 Halide perovskites in memory devices",level:"2"},{id:"sec_9_2",title:"4.3 Layered perovskites in memory devices",level:"2"},{id:"sec_11",title:"5. Design challenges of perovskites based memory devices",level:"1"},{id:"sec_12",title:"6. Conclusions",level:"1"},{id:"sec_13",title:"Acknowledgments",level:"1"}],chapterReferences:[{id:"B1",body:'Kamiya K, Yang MY, Magyari-Köpe B, Nishi Y, Shiraishi K. 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Journal of Materials Chemistry C. 2020;8:12865-12875. DOI: 10.1039/D0TC02579H'},{id:"B62",body:'Paramanik S, Maiti A, Chatterjee S, Pal AJ. Large resistive switching and artificial synaptic behaviors in layered Cs3Sb2I9 lead-free perovskite memory devices. Advanced Electronic Materials. 2022;8:2100237. DOI: 10.1002/aelm.202100237'},{id:"B63",body:'Park Y, Lee J-S. Controlling the grain size of Dion–Jacobson-phase two-dimensional layered perovskite for memory application. ACS Applied Materials & Interfaces. 2022;14:4371-4377. DOI: 10.1021/acsami.1c20272'},{id:"B64",body:'Tian Q , Hong R, Liu C, Hong X, Zhang S, Wang L, et al. Flexible SnO optoelectronic memory based on light-dependent ionic migration in Ruddlesden–Popper perovskite. Nano Letters. 2022;22:494-500. DOI: 10.1021/acs.nanolett.1c04402'},{id:"B65",body:'Gedda M, Yengel E, Faber H, Paulus F, Kreß JA, Tang M-C, et al. Ruddlesden–Popper-phase hybrid halide perovskite/small-molecule organic blend memory transistors. Advanced Materials. 2021;33:2003137. DOI: 10.1002/adma.202003137'},{id:"B66",body:'Li J, Tang X-G, Liu Q-X, Jiang Y-P, Li W-H, Tang Z-X. Interfacial resistive switching properties of Sr2TiO4/SrTiO3 heterojunction thin films prepared via sol-gel process. Ceramics International. 2021;47:18808-18813. DOI: 10.1016/j.ceramint.2021.03.216'},{id:"B67",body:'Solanki A, Guerrero A, Zhang Q , Bisquert J, Sum TC. Interfacial mechanism for efficient resistive switching in Ruddlesden–Popper perovskites for non-volatile memories. Journal of Physical Chemistry Letters. 2020;11:463-470. DOI: 10.1021/acs.jpclett.9b03181'},{id:"B68",body:'Tian H, Zhao L, Wang X, Yeh Y-W, Yao N, Rand BP, et al. Extremely low operating current resistive memory based on exfoliated 2D perovskite single crystals for neuromorphic computing. ACS Nano. 2017;11:12247-12256. DOI: 10.1021/acsnano.7b05726'},{id:"B69",body:'Faraz A, Deepak N, Schmidt M, Pemble ME, Keeney L. A study of the temperature dependence of the local ferroelectric properties of c-axis oriented Bi6Ti3Fe2O18 Aurivillius phase thin films: Illustrating the potential of a novel lead-free perovskite material for high density memory applications. AIP Advances. 2015;5:087123. DOI: 10.1063/1.4928495'},{id:"B70",body:'Keeney L, Maity T, Schmidt M, Amann A, Deepak N, Petkov N, et al. Magnetic field-induced ferroelectric switching in multiferroic Aurivillius phase thin films at room temperature. Journal of the American Ceramic Society. 2013;96:2339-2357. DOI: 10.1111/jace.12467'},{id:"B71",body:'Faraz A, Maity T, Schmidt M, Deepak N, Roy S, Pemble ME, et al. Direct visualization of magnetic-field-induced magnetoelectric switching in multiferroic aurivillius phase thin films. Journal of the American Ceramic Society. 2017;100:975-987. DOI: 10.1111/jace.14597'},{id:"B72",body:'Panda D, Tseng TY. Perovskite oxides as resistive switching memories: A review. Ferroelectrics. 2014;471:23-64. DOI: 10.1080/00150193.2014.922389'},{id:"B73",body:'Ye H, Sun B, Wang Z, Liu Z, Zhang X, Tan X, et al. High performance flexible memristors based on a lead free AgBiI4 perovskite with an ultralow operating voltage. Journal of Materials Chemistry C. 2020;8:14155-14163. DOI: 10.1039/D0TC03287E'},{id:"B74",body:'Gu C, Lee JS. Flexible hybrid organic-inorganic perovskite memory. ACS Nano. 2016;10:5413-5418. DOI: 10.1021/acsnano.6b01643'},{id:"B75",body:'Hwang B, Lee J-S. Hybrid organic-inorganic perovskite memory with long-term stability in air. Scientific Reports. 2017;7:673. DOI: 10.1038/s41598-017-00778-5'},{id:"B76",body:'Han JS, Van LQ , Choi J, Kim H, Kim SG, Hong K, et al. Lead-free all-inorganic cesium tin iodide perovskite for filamentary and interface-type resistive switching toward environment-friendly and temperature-tolerant nonvolatile memories. ACS Applied Materials & Interfaces. 2019;11:8155-8163. 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Centre for Advanced Devices and Systems, Faculty of Engineering, Multimedia University, Malaysia
'},{corresp:null,contributorFullName:"Mohd Arif Mohd Sarjidan",address:null,affiliation:'
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Department of Chemical and Petroleum Engineering, United Arab Emirates University, United Arab Emirates
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The Open Access Publishing Fee (OAPF) is payable only after your book chapter, monograph or journal article is accepted for publication.
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OAPF Publishing Options
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1,400 GBP Chapter - Edited Volume
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850 GBP Chapter - Book Series Topic (Annual Volume)
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10,000 GBP Monograph - Long Form
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4,000 GBP Compacts Monograph - Short Form
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850 GBP Journal Article (Across Portfolio)
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During the launching phase journals do not charge an APC, rather they will be funded by IntechOpen.
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Services included are:
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An online manuscript tracking system to facilitate your work
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Personal contact and support throughout the publishing process from your dedicated Author Service Manager
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Assurance that your manuscript meets the highest publishing standards
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English language copyediting and proofreading, including the correction of grammatical, spelling, and other common errors
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XML Typesetting and pagination - web (PDF, HTML) and print files preparation
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Discoverability - electronic citation and linking via DOI
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Permanent and unrestricted online access to your work
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What isn't covered by the Open Access Publishing Fee?
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If your manuscript:
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If a manuscript requires Heavy Editing or Language Polishing, this will incur additional fees.
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Your Author Service Manager will inform you of any items not covered by the OAPF and provide exact information regarding those additional costs before proceeding.
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Open Access Funding
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To explore funding opportunities and learn more about how you can finance your IntechOpen publication, go to our Open Access Funding page. IntechOpen offers expert assistance to all of its Authors. We can support you in approaching funding bodies and institutions in relation to publishing fees by providing information about compliance with the Open Access policies of your funder or institution. We can also assist with communicating the benefits of Open Access in order to support and strengthen your funding request and provide personal guidance through your application process. You can contact us at funders@intechopen.com for further details or assistance.
\n\n
For Authors who are still unable to obtain funding from their institutions or research funding bodies for individual projects, IntechOpen does offer the possibility of applying for a Waiver to offset some or all processing feed. Details regarding our Waiver Policy can be found here.
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Added Value of Publishing with IntechOpen
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Choosing to publish with IntechOpen ensures the following benefits:
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Indexing and listing across major repositories, see details ...
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Long-term archiving
\n\t
Visibility on the world's strongest OA platform
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Live Performance Metrics to track readership and the impact of your chapter
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Dissemination and Promotion
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Benefits of Publishing with IntechOpen
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Proven world leader in Open Access book publishing with over 10 years experience
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+5,700 OA books published
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Most competitive prices in the market
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Fully compliant with OA funding requirements
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Optimized processes that assure your research is made available to the scientific community without delay
\n\t
Personal support during every step of the publication process
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+184,650 citations in Web of Science databases
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Currently strongest OA platform with over 175 million downloads
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