The human LINE-1 (or L1) element is a non-LTR retrotransposon that is mobilized through an RNA intermediate by an L1-encoded reverse transcriptase and other L1-encoded proteins. L1 elements remain actively mobile today and continue to mutagenize human genomes. Importantly, when new insertions disrupt gene function, they can cause diseases. Historically, L1s were thought to be active in the germline but silenced in adult somatic tissues. However, recent studies now show that L1 is active in at least some somatic tissues, including epithelial cancers. In this review, we provide an overview of these recent developments, and examine evidence that somatic L1 retrotransposition can initiate and drive tumorigenesis in humans. Recent studies have: (i) cataloged somatic L1 activity in many epithelial tumor types; (ii) identified specific full-length L1 source elements that give rise to somatic L1 insertions; and (iii) determined that L1 promoter hypomethylation likely plays an early role in the derepression of L1s in somatic tissues. A central challenge moving forward is to determine the extent to which L1 driver mutations can promote tumor initiation, evolution, and metastasis in humans. Transposable genetic elements constitute at least 45% of the human genome . Some ofsequence ,4. The eIn order to examine how L1s influence human genomes and disease, we must first understand how these elements are mobilized .cis preference for mobilizing the specific mRNA that gave rise to them, which allows for the preferential amplification of functional L1 copies [This process begins with a canonical full-length L1 (FL-L1) source element that is (A) tail A. Only apromoter A, most litiation . ORF2p iitiation  and reveitiation . These pitiation ,11 that 1 copies ,13. Receng genes . Althougng genes .Bombyx mori [The final step of L1 mobilization is known as target-primed reverse transcription (TPRT) F. This mbyx mori , and latbyx mori . BrieflyTTT/A-3′ ,17,18, aTTT/A-3′ . This cl(A) tail ,21 and ethe mRNA ,16. Finathe mRNA .Alu [New L1 insertions have important hallmark features of retrotransposition: a poly(A) tail, flanking target site duplications (TSDs), frequent 5’ truncation, and occasional 5’ inversion ,24,25,26Alu  and SVA Alu ,32, as wAlu . As a coAlu ,34.The large number of individual steps that are required for L1 mobilization present numerous opportunities for human cells to regulate this mutagenic activity, and many such L1 regulators have evolved. In fact, there are mechanisms of L1 repression that act at each step of the mobilization process. For example, one of the most well-studied mechanisms of L1 repression is the methylation of CpG dinucleotides in the L1 promoter ,36. PromThe Selfish Gene in 1976, and there were even ongoing reviews and debates about this notion in Nature in 1980 [Mobile genetic elements were first discovered in maize genomes by Barbara McClintock in 1950 . This wo in 1980 ,45,46. H in 1980  and by oDuring the 1980s there was a furious rush to describe the newly-discovered L1 repetitive elements. This included classifying L1s, determining their function, and beginning to understand their activity—all of which occurred in the short span of eight years. When L1s were first discovered in 1980, they were simply described as long repeated sequences of variable length that were dispersed across all human chromosomes . ResearcF8) on Chromosome X. Two unrelated patients were found to have germline L1 insertions in the F8 gene that were absent from their parents’ genomes, indicating that these L1 insertions probably occurred in gametes or during early embryogenesis [MYC proto-oncogene. However, only part of this L1 insertion was sequenced and the structure lacked the hallmarks of TPRT [The first evidence for ongoing mobility of L1s in human genomes came in 1988, when Kazazian et al. found two disease-causing de novo germline L1 insertions . This L1ogenesis . The twoogenesis . This 7– of TPRT . Thus, aF8 [APC tumor suppressor gene (TSG) in tumors of CRC patients. This screen was performed because APC plays a pivotal role in this kind of cancer: the majority of CRC tumors are initiated by mutation or loss of function of both APC alleles in normal colon cells, which results in the formation of a precancerous lesion that can eventually progress to carcinoma [APC, so it was expected to disrupt normal APC function. The authors emphasized that the insertion likely initiated the tumor, but in view of our current understanding of CRC, this interpretation is somewhat unclear  . SimilarF8 , this inarcinoma ,54,55. TAlu, SVA, or other mRNAs (reviewed in [For many years this was the only available evidence that L1 elements were capable of retrotransposition in somatic tissues. There was another putative example of somatic L1 activity published in 1997 . Howeveriewed in ). In mosAlu [At this point in time scientists were undertaking important mechanistic research on L1 retrotransposons, including studies aimed at understanding how L1s are mobilized .As outlined above in As mentioned above, we rediscovered somatic L1 retrotransposition in human tumors in a study that leveraged next-generation sequencing technologies to investigate L1 retrotransposition in human populations and cancers . This stAfter developing and optimizing the L1-Seq approach, we used it to discover new L1 insertions in 38 diverse humans, eight tumor-derived cell lines, 20 non-small cell lung tumors , and 10 brain tumors (with matched blood leukocyte controls) . This scDuring the seven years that have elapsed since our 2010 study , many adAlthough these studies employed several strategies to measure somatic L1 activity, most of the methods can be grouped into two basic categories: (1) targeted resequencing assays and (2) bioinformatics tools that use whole genome sequencing (WGS) or whole exome sequencing (WES) to discover somatic L1 insertions. Targeted resequencing tools exploit specific sequence features of young L1s to selectively amplify and sequence novel L1 insertion junctions. These assays are similar to the previously described L1 Display  and L1-SSomatic L1 activity in cancer genomes has been found to be almost entirely confined to tumors arising from epithelial tissues  see Tab. A similer tumor .Additionally, L1s are responsible for the occasional somatic retrotransposition of other sequences in cancer genomes. The most commonly mobilized non-L1 sequences are 3ized see . In one 24% 655/26 of the 24% 655/26 of the n tumors ,88. Thes24% 655/26 of the Alu insertions also have been observed in various epithelial cancers [Processed pseudogenes also are generated in somatic tissues via the retrotransposition of cellular mRNAs by the L1 machinery ,90. Such cancers ,71,74,75It is increasingly becoming possible to identify the specific FL-L1 source elements that produce somatic offspring insertions in tumors. A key development in this regard has been a much more extensive knowledge of the FL-L1 source elements that are harbored by human genomes. Based on the reference (REF) human genome sequence, Brouha et al. initially estimated that every individual has a collection of approximately 80 to 100 FL-L1 source elements that are retrotransposition-competent . Later sCurrently, there are two available methods to determine source/offspring relationships between L1 elements. The first uses 3ductions ,73,75. Wductions . Each soductions .APC TSG and initiated tumorigenesis [Both of these methods have been used to study the FL-L1 elements that are active in human tumors. One large study employed the 312 types . Even moductions . Activitductions ,75. Thise of CRC . Three nigenesis .These studies have uncovered some important features of active source elements. For instance, both REF and non-REF FL-L1 source elements can contribute to somatic L1 retrotransposition ,80. SourThe discovery of frequent somatic L1 activity in cancer has researchers asking why this phenomenon is occurring and why it is so variable between tumors and tissues. Promoter methylation is one of the earliest lines of defense against L1 activity, and therefore, hypomethylation is thought to be a necessary step for L1 reactivation in tumors. This hypothesis has been corroborated by many groups. These studies examined methylation at three different levels, using techniques that vary in scope. First, the Illumina Infinium platform assesses global genomic methylation within CpG islands . Second, primer) ,80.Using the first method (the Illumina Infinium platform), we identified a hypomethylation signature at 59 genomic CpG sites that was associated with L1-permissive lung cancers . Soon thIn addition to cancer genomes, somatic retrotransposition also occurs in neuronal cells ,85 and nAPC TSG and likely was a driver of tumorigenesis [APC allele was not examined in that study and the authors did not rule out a possible role for faulty DNA repair as an initiator of tumorigenesis [PTEN . It canscovered ,75.One major unresolved question is: how often do L1 driver mutations initiate tumorigenesis in normal cells? Insertions that occur after tumor growth is well underway would not appear to participate in tumorigenesis per se, but instead might play a role in tumor evolution or metastasis. Doucet-O’Hare et al. examined the evolution of esophageal cancer from a precancerous condition and demonstrated that L1s can be active very early during the tumorigenesis process, and even found somatic L1 insertions in precancerous lesions that never progressed to cancer (over a 15 year period) . In a siAPC is a gatekeeper TSG that is frequently mutated in patients with CRC—in fact, both copies of APC must be mutated to initiate most cases of CRC [APC allele was inactivated by a point mutation, and that this tumor had stable microsatellites, indicating that faulty DNA repair did not initiate tumorigenesis in this case [APC gatekeeper alleles are mutated in a normal cell [APC alleles and a stop codon disrupted the second allele. Thus, somatic L1 mutations can initiate tumorigenesis in normal colon cells.We recently demonstrated that L1 indeed can initiate tumorigenesis in normal colon cells . APC is s of CRC ,54,55. Ws of CRC , only 38s of CRC  . Even cancer ).Another confounding factor is that the temporal order in which gene mutations occur during tumor initiation and evolution is unclear in most tumor types (recently reviewed in ). In manAPC occurred at the earliest stages of tumorigenesis , providing further evidence that normal colon tissues can support somatic L1 activity [Although L1 clearly is quite active in the normal somatic tissues of the brain and epithelial cancers, we are just beginning to explore the extent of somatic activity in other normal tissues. Several lines of evidence suggest that L1 can evade somatic repression in at least some other normal tissues. In one study, 21 putative somatic L1 insertions were identified in normal liver cells using a targeted sequencing assay . In anotactivity . Thus, tOn the basis of this limited evidence, is it possible that most  normal epithelial tissues support L1 activity? If so, this might have been largely missed for the same reason that it was initially overlooked in the brain: that each cell in a given tissue generates a unique collection of somatic L1 retrotransposition events that cannot be detected in bulk tissue. The solution to this problem is to adapt L1 discovery methods to single cell sequencing technologies. This approach has been pioneered in brain tissues by the Walsh  and FaulFinally, we need to address how differences in FL-L1 source element profiles influence tumorigenesis. As discussed in We also need to gain a better understanding of how FL-L1 source element profiles vary in human populations. Although some FL-L1s are ubiquitously found in most or all human genomes, many others are found only in a subset of individuals and are inherited in a population-specific manner . As a reWe have presented a brief overview of research examining somatic L1 retrotransposition in human genomes, focusing on landmark studies outlining the activity of these mobile elements in human cancers. Researchers in this field have characterized many aspects of somatic L1 activity in the short span of only seven years. However, several major questions remain unresolved. One unsettled question is: How often does somatic L1 retrotransposition initiate and drive tumorigenesis in humans? Despite the fact that thousands of somatic L1 insertions have been recovered from many tumor types, only a handful of clearly-recognizable driver mutations have been discovered. We also have a very incomplete understanding of L1 activity in normal somatic tissues. If L1s are highly active in most adult somatic tissues, we clearly need to gain a better understanding of the consequences of this activity. Likewise, in our current era of human genomics and precision medicine, it is increasingly important to define inter-individual variation and determine how variation in FL-L1 source element profiles may impact human health and disease.


The exceptional properties of graphene make it a promising candidate in the development of next‐generation electronic, optoelectronic, photonic and photovoltaic devices. A holy grail in graphene research is the synthesis of large‐sized single‐crystal graphene, in which the absence of grain boundaries guarantees its excellent intrinsic properties and high performance in the devices. Nowadays, most attention has been drawn to the suppression of nucleation density by using low feeding gas during the growth process to allow only one nucleus to grow with enough space. However, because the nucleation is a random event and new nuclei are likely to form in the very long growth process, it is difficult to achieve industrial‐level wafer‐scale or beyond (e.g. 30 cm in diameter) single‐crystal graphene. Another possible way to obtain large single‐crystal graphene is to realize ultrafast growth, where once a nucleus forms, it grows up so quickly before new nuclei form. Therefore ultrafast growth provides a new direction for the synthesis of large single‐crystal graphene, and is also of great significance to realize large‐scale production of graphene films (fast growth is more time‐efficient and cost‐effective), which is likely to accelerate various graphene applications in industry. Pt is one of the promising substrates, while at the moment it still cannot meet the demands for the large‐scale synthesis of graphene. Ni and Cu are much cheaper and widely available substrates that make the industrialized production of graphene feasible. High solubility of carbon in Ni leads to the formation of multilayer graphene.Graphene, a two‐dimensional material with carbon atoms bonded in a honeycomb lattice, has attracted immerse interests in the past decade due to its exceptional properties and various applications.However, as‐synthesized graphene films on Cu foils are typically polycrystalline and the grain boundaries degrade the properties greatly, limiting its high‐end applications.2Figure−1. That means to grow 12‐inch single‐crystal graphene films typically needs almost one day. This time‐ and energy‐consuming process is not suitable for the large‐scale production. Only from 2016, several breakthroughs made the growth rate exceed 100 µm min−1,−1.FigureAfter about a decade's efforts, the domain size of single‐crystal graphene has increased more than four orders of magnitude, from micrometres to inches Figure2.31, 334 makes it a preferred carbon source to prevent pyrolysis at high temperatures, which is favourable to surface‐mediate growth of high‐quality graphene.4 onto Cu surfaces; (ii) catalytic dissociation of CH4 by Cu, resulting in C species CHx (x = 0–3); (iii) surface diffusion of C species; (iv) C attachment onto graphene domain edges. A large number of experiments have shown that these four processes in graphene growth are dominated by the catalytic substrate, feeding gas, reaction barriers, temperature and pressure  that is catalytically decomposed from CH4 and absorbed on the Cu surface, increasing active carbon on Cu surface can directly enhance the graphene growth rate. However, the high carbon species concentration on the Cu surface also leads to more nucleation and thus smaller graphene domain sizes. People desire large graphene domains and try to have little active carbons on Cu surface to suppress the nucleation density with the compromise of growth rate. That is the reason why the reported growth rate is usually very low on Cu. It seems to be a dilemma in optimizing both the domain size and growth rate. A good way to get out of this dilemma is to use Cu‐Ni alloy substrate. Ni has higher catalytic activity and carbon solubility than Cu and can be alloyed with Cu with any element content, thus changing the growth behaviors.The metallic substrate in the CVD graphene growth also serves as catalyst for the CH4 would have high colliding frequency and substantially enhance the local concentration of carbon flux, and the hence graphene growth rate is improved.Besides the composition, the configuration of substrate also affects the growth rate distinctly. When rolling or vertically stacking Cu foils via direct physical contact with a gap of about tens of micrometres, the gas flow in the narrow spaces of the stacked Cu foils could be in the molecular flow regime, where the CHAlso the surface properties, including surface index, step, bunches, impurities and defects, have dramatic influence on the graphene growth. Generally, Cu foils used to grow graphene are polycrystalline and on different facet surfaces graphene growth behaviours are different. The growth rate on Cu(111) is higher than (100) and other high index facets, attributed to higher diffusion and improved absorption of hydrocarbon precursor.3.24 is typically used as the carbon source for graphene growth and thus can affect the growth rate directly. Apparently a higher concentration of CH4 would lead to faster growth of graphene over the Cu surface, while it also results in higher nucleation density. The nucleation occurs when the C species on the active sites are oversaturated and if the CH4 flow is too low that there will be no nucleation and growth.4 feeding, namely low feeding at the nucleation stage and high feeding in the growth stage, to achieve lower nucleation density but large graphene domain size.CH2 also plays vital roles in the graphene growth process. Actually, Cu can catalyse hydrogen dissociation reaction as well.2 would reduce surface sites, leaving fewer available Cu sites for CHx (x = 1–4) dissociative chemisorption. In addition, the dissociated H atoms are active to etch the edge carbon atom of existing graphene domains.2 is impeditive to the rapid growth, it is essential to achieve monolayer growth.HFigureAs discussed above, growth process is a balance between carbon diffusion/deposition and hydrogen etching , is thermodynamically unfavourable and the reaction barrier is relative high.x–1) may combine with each other and finally form H‐terminated graphene domains. Thus further C species edge attachment and lattice incorporation for the graphene domain growing up require dehydrogenation. In a typical graphene growth process, the growth rate can be either diffusion‐limited (insufficient active C supply) or edge‐attachment‐limited.In the growth process, the dissociation of CH4 via the reaction CHx + O → Cu  CHx–1 + OH . As shown in Figure4 decomposition, the O atom on the Cu surface is able to interact with the H atom by the formation of O–H bond3 radicals by several orders of magnitude. Considering that a CH4 molecule decomposition is a complicated process, we carefully evaluated the full dehydrogenation of CH4 by Climbing Image‐Nudged Elastic Band (CI‐NEB) calculation and the result is presented in Figure 3, CH2, CH and C, on the metal surface and/or in the gas phase. Thus, no matter which kind of C species should be the key participant of the graphene growth, a quick supply of C source around each graphene domain is guaranteed.It was reported the O on Cu surface can reduce the energy barrier to the dissociation of CHAlso, given that the H on the Cu has lower energy in the form of OH compared with that of H on Cu3.44 is chemically stable. Therefore, the temperature must be much higher using Cu as substrate and CH4 as carbon source. Usually, 1000 °C or above is applied to achieve the growth of high‐quality single‐crystal graphene. At lower temperature, like 800 °C, graphene can be synthesized but with poor quality.4, diffusion of active carbon and attachment of C species onto graphene nuclei. Also, higher temperature can decrease the nucleation density of graphene.As an active factor in the CVD growth of graphene, temperature can affect the growth rate significantly. The catalytic activity of Cu is lower compared to other mentioned metal catalyst and CH4 on Cu surface is enhanced.4.4 flux is high, the self‐limited growth process would be broken and multilayer graphene may form.4 is not a good choice for the growing of high‐quality graphene.Pressure is another important factor in the growth process. It has been widely accepted that low pressure CVD can readily achieve uniform monolayer graphene film. At lower pressure, the lower gas flux results in fewer collisions and a higher diffusivity coefficient, and thus the absorption of CHBased on the above discussion, the effects of temperature and pressure on growth rate are interactive. Appropriate temperature and pressure conditions can be reached to produce graphene with high growth rate and good quality.4To improve the growth rate of CVD graphene is essential for both fundamental research and industrial applications. In this review, the available ways towards the ultrafast graphene growth are discussed in details. Proper catalytic substrate can achieve the higher growth rate, while it is more powerful in the control of layer number. The optimization of feeding gas ratio has limited capacity to improve the grow rate. The deduction of reaction barrier, for example by the introduction of oxygen, can improve the growth rate exponentially and turns out to be a very powerful way to realize ultrafast growth.In the future, further work is required for faster growth and larger graphene single crystals. Several possible routes are proposed: (i) to explore a more efficient way to reduce the reaction barrier for the graphene growth; (ii) to epitaxially grow well aligned graphene domains and make them seamlessly stitch together into a complete single‐crystal film; (iii) to combine with roll‐to‐roll technique to realize streamline production. Route (i) can be realized by effective introduction of oxidative substance like O onto Cu surface, to enhance the dissociation of hydrocarbon. This is expected to readily obtain millimetre‐scale single‐crystal graphene islands in several minutes. Actually, route (ii) has been implemented by growing graphene on Ge(110)The authors declare no conflict of interest.


In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous HPV infections.Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17–36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV.  Papillomaviruses (PV) are a diverse group of non-enveloped, double-stranded, species-specific DNA viruses with strict epithelial tissue tropism. More than 200 human PV (HPV) genotypes have been completely sequenced and classified into five genera: alpha, beta, gamma, mu and nu  . While gA group of HPV types belonging to genus beta are hypothesized to play a role as adjunct to the main carcinogen UV light in the development of non-melanoma skin cancers (NMSC) in immunosuppressed OTR and HIV-positive patients. Although beta HPV are detected in skin swabs or hair bulbs from normal individuals by highly sensitive nested PCR, infections appear to be contained by the immune system. Beta HPV have originally been identified in patients with the rare genodermatosis Epidermodysplasia verruciformis (EV), who typically present with generalized flat warts and scaly plaques. Early in life EV patients develop multiple NMSC, squamous cell carcinomas (SCC) or basal cell carcinomas (BCC), predominantly on sun-exposed areas . The genWhile the role of beta HPV causing NMSC in EV patients is long established, their role in development of NMSC in OTR is less clear. Beta HPV infections of the skin are acquired very early in life, even a few days after birth, reaching an overall prevalence of approximately 90% with increasing age without causing clinical symptoms. Studies have revealed increased beta HPV DNA carriage in cutaneous SCC compared to perilesional skin, whereas prevalence and viral load appear higher in precancerous actinic keratoses, opposite to the findings in EV patients –14. In c®), HPV16/18/6/11 , or HPV16/18/31/33/45/52/58/6/11 , with the potential to protect against 70–90% of all CxCa cases and, for the latter two vaccines against 90% of genital warts. However, they do not protect against the mucosal types associated with the remaining 10% of CxCa cases requiring cervical screening programs to be maintained in vaccinated women  and CsCl-PBS [29% (wt/wt)] density gradients .Viral fusion proteins were identified by SDS-PAGE / Coomassie staining or Western Blot using mAb Camvir-1   or a HPV2 +0.02% Tween 80. Fifty μg of respective chimeric VLP were adjuvanted with 500μg alum plus 50μg MPL  and used to immunize two NZW   each in a 5-dose regimen at week 0-3/4-6-8-16 for HPV1L1-4RG1 VLP, or in a 4-dose regime at week 0-4-6-8 for HPV16L1-17RG1, HPV5L1-17RG1, 16L1-5L2(aa53-72) or 18L1-5L2(aa53-72) VLP, and sera drawn two weeks after the final boost.Gradient-purified proteins were extensively dialyzed against PBS +0,5M NaCl +1mM CaCl3 (pH 10.6) +0.01M freshly added dithiothreitol (DTT) buffer over night at 37°C. The next day wells were washed, blocked with 0.5% milk/PBS, and serial dilutions of antibodies  added in triplicates for one hour at room temperature (RT). Antibodies used were neutralizing anti HPV16 L1 mouse mAb H16.V5, H16.5A, H16.14J, 263.A2, H16.E70, or neutralizing anti HPV18 L1 mAb H18.J4 , or non-neutralizing mAb Camvir-1 directed to a linear HPV16 L1 epitope (BD Pharmingen), or rabbit serum raised against HPV16 L2 aa11-200 . Chimeric HPV5L1-17RG1 and HPV1L1-4RG1 VLP were evaluated using the HPV16L2 aa11-200 polyclonal serum as no Ab to HPV1 or HPV5 L1 were available. After a PBS wash step, the second antibody goat anti-mouse or goat anti-rabbit IgG-HRP (Bio-Rad) was added at 1:10,000 dilution for 45 minutes at RT, followed by substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (Roche). The OD at 405nm was determined with an ELISA reader  and positive titers are reported for OD values greater than the mean OD of pre-immune sera plus 3 standard deviation (SD).Antigenicity of chimeric L1-RG1 or L1-L2(aa53-72) VLP as compared to wt HPV16L1 or wt HPV18L1/L2 VLP was analyzed by ELISA. VLP preparations were quantified by SDS-PAGE Coomassie staining by visually comparing the intensity of the L1 band at ~50 kDa to serial 1:2 dilutions (from 1 mg/ml to 0.125 mg/ml) of a bovine serum albumin standard (possible impurities or degraded L1 proteins were not taken into account). In short, 0.1μg native VLP per well in 100μl PBS were attached onto Maxisorp 96-well plates (Nunc) over night at 4°C. For denaturing conditions, 0.1μg VLP were dried onto the ELISA plate without a lid in 0.2M NaHCOTo evaluate immunogenic display of the genetically inserted L2 epitopes on the VLPs’ surface, peptide ELISAs were performed using biotinylated RG1-homology peptides of either HPV17 or HPV4, or the HPV5 L2 epitope aa53-72 . Briefly, 1μg biotinylated peptide in 100μl coating buffer (1M Tris/HCl (pH7.4) +1M NaCl +0.001% Tween-20) per well were plated at 4°C overnight onto Streptavidin plates (Nunc immobilizer Streptavidin F96 clear). Wells were then washed with coating buffer for 5 minutes and blocked with 1% milk/PBS at 4°C overnight, washed again and incubated in triplicates with serial dilutions of antisera  for one hour at RT on an ELISA plate shaker. Pre-immune or immune sera raised against wt HPV18L1, HPV16L1, HPV5L1 VLP, or a human HPV1-reactive serum were used as controls. The second antibody goat anti-rabbit IgG-HRP (Bio-Rad) was added for 45 minutes at dilution 1:10,000 at RT, prior to adding the substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (Roche). The OD at 405nm was determined as stated above and titers are reported for values greater than the mean OD of pre-immune sera plus 3 standard deviation (SD).https://home.ccr.cancer.gov/Lco/codonmodification.htm), further edited by hand, synthesized  and sub-cloned into the double expression vector pVitro1-neo-mcs (Invivogen). The BamHI-XbaI flanked L1 was cloned into MCS2 using the BglII-NheI sites, while the BamHI-XbaI flanked L2 gene was inserted into MCS1 using the BamHI-AvrII sites , HPV17 , HPV20 (U31778), HPV23 (U31781), HPV24 (U31782), HPV36 (U31785), HPV37 (U31786), HPV49 (X74480), HPV80 (Y15176), HPV92 AF531420), HPV93 (AY382778) and HPV96 (AY382779) were designed according to published instructions . L1 and 1420, HPV6 293TT cells were plated into 175cm2 flasks and one day later transfected with 38μg DNA each of HPV L1 and L2 expression and reporter plasmids . On the next day media was changed, and 48 hours after transfection cells were lysed in PBS +9.5mM MgCl2 +0.5% volume Triton-X100 +0.25% Ammonium sulfate +0,1% Benzonase (of a 25U/μl stock) +0,1% Plasmid Safe. For particle maturation cell lysates were incubated 20–24 hours at 37°C. Finally, PsV were salt extracted and purified by ultracentrifugation on Optiprep step gradients .PsV were produced in 293TT cells as described , 55. BriPlasmids for L1 and L2 of HPV16 and HPV18 were provided by J. Schiller, NCI; for HPV5 and 45 by C. Buck, NCI; for HPV3 and 76 by J. Dillner, Karolinska Institute; for HPV27 and 57 from ; and forExpression of L1 and L2 proteins of novel beta PsV was verified by SDS-Coomassie staining and Western Blot using either mAb Camvir-1 (BD Pharmingen) , mAb AU1In order to generate type-specific control immune sera, one NZW rabbit each was immunized with HPV4, 20, 24, 36, 38, 39, 59, 68, 76 and 96 in a 3-dose regime at week 0-3-6 using 20μg PsV and Freund’s adjuvants (CFA/IFA) and sera finally drawn two weeks after the final boost (Charles River Laboratories). Additionally, HPV15, 23, 37, 49 and 80 PsV were used to immunize groups of mice (n = 5) subcutaneously with 50ng PsV plus alum  as adjuvant in a 2-dose regime (week 0–2) and sera were drawn and pooled two week after the final boost.®, Life Technologies), subcloned into pET28a vector and, and used to transform Rosetta DE3 cells (Novagen). Protein expression was induced by IPTG  as per company’s instructions and purified via its His-tag using NTA columns . 300μg peptide adjuvanted with CFA/IFA were used to immunize two NZW in a 3-dose regime at week 0-3-6, and final immune sera drawn two weeks after the final immunization.A gene encoding 17RG1x5 peptide  was synthesized (GeneArt4 293TT cells per well were plated onto a 96-well plate and infected with SEAP-containing PsV that were pre-incubated without or with (pre-) immune sera on ice, for one hour. Infection was assessed three days later by measuring the SEAP signal at 405nm  after addition of 4-nitrophenyl phosphate disodium salt hexahydrate  dissolved in diethanolamine (Sigma). Additionally, neutralization assays of types that showed low infectivity  were evaluated using Ziva Ultra SEAP Plus Kit (Jaden BioScience) or GreatEscape SEAP chemiluminescence kit  according to company’s instructions. In short, after 72 hour incubation, plates were shaken for a minute to gain a homogenous supernatant prior centrifugation for 5 minutes at 1700xg. 5 or 20μl were then transferred onto an opaque 96-well Optiplate (Nunc) containing 75μl 1x SEAP Sample Preparation Solution. Plates were shaken again and incubated at 65–68°C for 50 minutes. Following this inactivation of endogenous SEAP, 25μl (for the ZIVA Ultra SEAP Plus Kit) or 100μl (for the GreatEscape SEAP Kit) of substrate were added and plates incubated in the dark for 30 minutes prior reading luminescence for 0.2–0.3 seconds per well using either the Victor3 or Fluoroskan Ascent FL plate reader. Serum dilutions showing at least a 50% reduction in SEAP signal compared to pre-serum were considered to be neutralizing.The L1-based assay was performed as previously described . Briefly6 HaCaT cells were plated onto 96-well plates for 24 hours at 37°C, cells were washed twice with PBS and lysed using PBS +0.5% Triton X-100 +20mM NH4OH for 5 minutes at 37°C. Following lysis, 100μl PBS were added and removed again for three times to gently wash the remaining extracellular matrix, and PsV in 120μl/well CHOΔfurin conditioned medium containing 5μg/ml heparin (Gilvasan Pharma) added. The ECM/PsV mix was incubated overnight at 37°C and the next day washed with PBS to remove non-attached PsV. Antibody serial dilutions were added and incubated at 37°C for at least 1 hour. After this incubation step, 8*103 PGSA-745 cells were directly added to the wells (50μl/well) and incubated at 37° for two days. For luciferase encoding PsV, assays were evaluated using the Luciferase Assay System (Promega #E1501) and the Cell Culture Lysis Reagent (Promega #E153A) according company’s instructions in 96-well opaque Optiplates (Nunc). Luciferase activity was measured using either the 1420 Victor3 Multilabel Counter (PerkinElmer) or the Fluoroskan Ascent FL (Thermo Scientific) plate reader, with 10 second per well reading time. For SEAP encoding PsV, assays were evaluated using the Ziva Ultra SEAP Plus Kit (Jaden BioScience) or GreatEscAPe™ SEAP Kit  as mentioned before. For CHOΔfurin conditioned medium, 1*106 cells were grown in 17ml media and supernatants harvested after incubation at 37°C for 4 days. Serum dilutions showing at least a 50% reduction in SEAP signal compared to pre-serum were considered to be neutralizing. PGSA-745 and CHOΔfurin cells were kindly provided by Patricia Day, NCI.The L2-based assay was performed according to Day et al . In shormg/ml; Promega; 10 minute exposure) and imaging by the IVIS system . Data were analyzed by the Living image software program by drawing uniform regions of interest (ROI) around the luminescence emitting genital area of each mouse to determine the average radiance within the ROI. A group of mice (n = 5) was challenge with CMC only to determine background radiance. Luciferase activity was measured as p/s/cm2/sr (average radiance).Six to eight weeks-old female Balb/c were obtained from Charles River Laboratory and the challenge experiments were performed according to Roberts et al. . BrieflyThe cutaneous challenge was performed according Wu et al. . BrieflyAnimal studies were approved by the ethics committee of the Medical University of Vienna and the Austrian Federal Ministry of Science and Research (BMWF-66.009/0173-1l/3b/2011) and animal care was in accordance to the guidelines of the Institute for Biomedical Research Vienna. The vaginal/cutaneous challenges were performed under Ketaminhydrochlorid/Xylazin anesthesia to minimize suffering. Animals were sacrificed by cervical dislocation.In vivo statistical analysis was performed using Graph Pad Prism to evaluate p-values between indicated groups .Chimeric L1-L2 fusion genes were designed that encode L1 capsid proteins of hr mucosal HPV16 or HPV18, hr cutaneous beta HPV5, or common cutaneous HPV1 L1 as scaffold to present L2 peptides via the DE surface loop. The HPV17 (17RG1) and HPV4 (4RG1) L2 peptides, comprising each N-terminal 20 aa14-33, were chosen based on homology to the highly conserved broad cross-neutralization epitope ‘RG1’ of HPV16 L2 . The HPVRecombinant baculoviruses were isolated by plaque purification, amplified, and fusion proteins expressed by infection of Sf9 cells and verified by SDS-PAGE. By Coomassie staining, the five fusion proteins migrated around 50kDa . AdditioLarge molecular weight complexes were purified from infected Sf-9 cell extracts on density gradients, negatively stained by uranyl acetate and analysed by transmission electron microscopy (TEM). Electron micrographs showed spherical particles around 50-60nm for chimeric HPV5L1-17RG1 (A), HPV16L1-17RG1 (B), HPV1L1-4RG1 (C), HPV16L1-5L2(aa53-72) (D) and HPV18L1-5L2(aa53-72) (E) fusion proteins , indicatChimeric VLP were further analysed by ELISA for antigenicity of the VLP scaffold and ability to present inserted L2 epitopes. Under native conditions, HPV16-neutralizing mAb H16.E70 did not bind and H16.V5 only weakly bound to chimeric HPV16L1-17RG1VLP, whereas both mAbs reacted with wt HPV16 L1 VLP as expected  left, whNext, HPV16L1-5L2(aa53-72) VLP were examined by ELISA and compared to wt HPV16 L1+L2 VLP using conformation-dependent HPV16-neutralizing mAb. Similar to above results, neutralizing mAb binding to native chimeric VLP was weak as compared to wt HPV16 L1+L2 VLP, indicating that epitope insertion interfered with mAb binding . As expeFor chimeric HPV1L1-4RG1 VLP mAb Camvir-1 showed a similar binding pattern by ELISA as seen for the other chimeric VLP, with stronger reactivity to denatured than native VLP , whereasTo generate the tools for evaluating the potential of chimeric VLP immunogens to protect against beta HPV infections, expression vectors for 12 beta HPV PsV types were generated using respective codon-optimized L1 and L2 genes. These comprised HPV15, 17, 20, 23, 24, 36, 37, 49, 80, 92, 93 and 96, representing beta HPV species 1–5 and included types identified in skin cancer samples. Following transfection into 293TT cells and PsV purification on density gradients, L1 plus L2 capsid proteins were verified by Western Blot using mAb to L1, or polyclonal sera raised against heterologous beta PsV types if mAb were non-reactive. MAb Camvir-1 recognized HPV92 and HPV17 L1 proteins (and HPV16 L1 VLP used as positive control) , whereasFurther, assembly of beta HPV L1+L2 proteins into capsids was assessed by TEM of negatively stained PsV preparations. While spherical structures were observed for HPV15, 17, 20, 23, 24, 36, 37, 49, 80, 92 and 96, no capsids were visible for HPV93 PsV preparations . Of notein vitro neutralization assays, and those with high in vitro infectivity were tested in vivo in vaginal mouse challenges. HPV17 and 93 proved to be non-infectious in vitro even though HPV17 (but not HPV93) preparations showed numerous and correctly sized virions by TEM  and HPV18L1-5L2(aa53-72), high neutralization titers were detected against homologous L1-scaffold HPV16  and HPV18  by L1-PBNA , no  were detected by L2-PBNA against HPV4 only (a PBNA for HPV1 is not available), but not against any of the tested common cutaneous  or beta HPV types tested  by L1-PBNA .in vitro analysis for cross-neutralization, one antiserum each to HPV5L1-17RG1 (serum #2), HPV16L1-17RG1 (serum #3) and HPV1L1-4RG1 VLP (serum #2) was further evaluated for in vivo cross-protection using the murine vaginal challenge model [Following ge model .Passive transfer of HPV5L1-17RG1 VLP-raised serum protected mice partially from experimental challenge with HPV20, 24 and 96  against any of the tested cutaneous HPV except HPV4, and thus in vivo challenge was performed with this type only. As can be seen in For sera induced by HPV1L1-4RG1 VLP vaccination, While the involvement of beta HPV in NMSC carcinogenesis in immunosuppressed patients is supported by epidemiological and experimental data, a possible role in keratinocyte skin cancer of the general population is less clear. Beta HPV DNA is more frequently detected in premalignant actinic keratosis than in NMSC and only a minority of cancer cells harbor viral DNA, supporting a possible role in initiation but not maintenance of the tumor phenotype .In a recent study using the natural Mastomys natalensis papillomavirus (MnPV) mouse model, prophylactic vaccination of naïve mice, and even therapeutic vaccination of previously infected animals protected against development of MnPV-associated skin cancer . TherapeWe have used the RG1 homologs of beta HPV or a common cutaneous type as type-common experimental vaccine antigens to more directly target cutaneous HPV, compared to a previous HPV16-based chimeric RG1-VLP (HPV16L1-16RG1) immunogen , 47. ThePreviously, expression of recombinant HPV L1 proteins in insect cells has shown that truncation of the nuclear targeting and DNA binding signals at the C-terminus of HPV16 and HPV18 L1 reduced incorporation of cellular DNA, whereas the capacity to assemble in immunogenic VLP was retained . In addiThe current standard approach to assess neutralizing antibodies, which are the main effectors of protection induced by L1 VLP vaccination, is by ‘L1- sensitive’ PsV-based neutralization assays (L1-PBNA). However, the L1-PBNA was shown to be less sensitive and thereby possibly underestimating the extent of L2-mediated cross-protective humoral responses. Consequently, a novel L2-based neutralization assay that detects L2-mediated neutralization (L2-PBNA) with higher sensitivity, as well as L1-targeted neutralization, was employed to analyse the (cross-) neutralization spectrum of chimeric VLP .in vitro which were not protective against HPV5 PsV challenge in vivo, while cross-neutralization was observed against additional 5 of 13 beta HPV by L2-PBNA. Nevertheless, mice passively immunized with sera raised against chimeric HPV5L1-17RG1 VLP were partially protected against experimental challenge with HPV20, 24 and 96 PsV, but not against HPV38 and 76. These results suggest that 17RG1 epitope insertion into and sequence replacement of the DE loop disrupted or sterically hindered major HPV5 L1 neutralization epitope(s) exposure in the chimeric HPV5L1-17RG1 VLP. Unfortunately, mAb to analyze chimeric and parental HPV5 VLP for type-specific neutralization epitopes were not available. However, the binding of HPV16-neutralizing mAb H16.V5 was recently mapped to 17 residues found in five intertwined loops of two neighboring L1 molecules, comprising the DE and HI loops of one L1 and the BC, DE and FG loops of an adjacent L1 [Unexpectedly, for antisera to HPV5L1-17RG1 VLP low type-specific neutralizing titers to homologous HPV5 were detected acent L1 , 67. Invacent L1 .in vivo protection against HPV16 PsV challenge, indicating the chimeric HPV16 VLP retained protective immunogenicity. RG1 epitope insertion at L1 positions 136/137 might have disrupted the DE loop involvement in the H16.V5 recognition site, while residual binding might be due to the remaining FG residues. MAb 263.A2 appears to have a similar binding pattern to that of H16.V5, with FG and HI loop residues recognized by both mAb defining a complex epitope recognized by a majority of human sera in response to natural HPV16 infection [In vivo experiments further confirmed immunogen-mediated (cross-) protection against challenge with HPV20, 96 and the homologous HPV16, while partial protection was observed against HPV5, 24 and 38.Neutralizing mAb H16.V5 has been shown to block 75% of reactive human sera with HPV16 VLP. Although reactivity of H16.V5 with HPV16L1-17RG1 VLP was rather weak, immune sera conferred complete type-specific nfection , 68. No nfection , 70. Of in vivo protection by murine vaginal challenge, because infection at the cutaneous site with beta HPV5, a type showing usually high in vivo infectivity, was much lower and more variable when compared to infection of the vaginal mucosal and thus did not allow robust analysis of vaccine efficacy  revealed that 12 C-terminal aa of its L2 protein were predicted to be missing (residues 525–536: SLKKRKRKRKYL) by comparison to the HPV17 L2 sequence entry from the HPV PAVE homepage . The missing sequence is highly conserved to the C-terminus of HPV16 L2 that contains a nuclear localization signal (aa454-462), a nuclear export signal (aa462-471) and a dynein-binding site  [Novel beta HPV PsV types were designed according to HPV prevalence in NMSC of both immune-suppressed and immune—competent patients, and to provide at least one type representative of different beta HPV species . In genel 40 aa) . MutatioThe second L2 epitope used in this study is the HPV5 L2 aa53-72 homolog of HPV16 L2 aa56-75. The latter epitope has originally been described to induce cross-neutralizing antibodies against mucosal hr types . More rein vitro and partially protected in vivo against HPV4 challenge, likely by targeting the homologous HPV4 RG1 epitope. However, antisera failed to cross-neutralize distantly related cutaneous alpha and beta HPV by PBNA, which is likely due to ≤50% RG1 sequence homology to tested cutaneous types . Antisera were neutralizing us types .In conclusion, chimeric VLP immunogens that display cross-neutralization L2-RG1 epitopes of cutaneous HPV types are evolving as a promising tool to develop next generation L2-based prophylactic vaccines with broad-spectrum protection against HPV types associated with skin pathologies in immunocompetent and immunosuppressed populations.S1 Fig(A) Reactivity of non-neutralizing mAb Camvir-1 and HPV16-neutralizing mAb H16.5A, H16.14J, 263.A2 with native or denatured HPV16L1-17RG1 VLP. Analysis of L2 epitope presentation by HPV16L1-17RG1 VLP (B) and HPV5L1-17RG1 VLP (C) using Camvir-1 and an antiserum raised to HPV16 L2 aa11-200 under native and denatured conditions.(TIF)Click here for additional data file.S2 Fig(A) HPV16L1-5L2(aa53-72) and (B) HPV18L1-5L2(aa53-72) VLP were compared to wt L1+L2 VLP under denatured conditions using indicated mAb or polyclonal serum. (C) Native or denature HPV1L1-4RG1 VLP were analyzed with Camvir-1 and antiserum to HPV16 L2aa11-200.(TIF)Click here for additional data file.S3 FigGroups of Balb/c (n = 10) were passively immunized with pre-immune serum and infected with 40μl HPV5 PsV at the skin of the belly or the vaginal mucosa as described in Materials. After subcutaneous or intravaginal addition of D-luciferin, bioluminescence was evaluated by in vivo imaging. Data from the vaginal challenge was taken from (TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 TableMAb Camvir-1 or AU1, or polyclonal sera raised against the indicated L1 and/or L2 antigens, were used to detect L1 and L2 expression of a panel of beta HPV PsV by Western blot. Results are summarized. (-), (~) and (+) indicate negative, weak, or positive immunoreactivity, respectively. Empty boxes indicate that no experiment was done.(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.S5 Table(TIF)Click here for additional data file.


In this paper, the periodic double-layer graphene ribbon arrays placed near a metallic ground plate coated by a dielectric layer are proposed and analyzed by the coupled-mode theory (CMT) to predict the perfect absorption response in the mid-infrared region. Numerical simulations of the finite-difference time-domain (FDTD) method confirm this effect and give the underlying physical origin. The anti-symmetric dipole-dipole coupling mode is supported by the double-layer graphene ribbons and acts as the electrical resonance to suppress the reflection, because of the impedance matching. The transmission from this system is restricted by the ultra-thick metallic ground plate. All incident electromagnetic energy is efficiently confined in the interlayer between graphene ribbons and the metallic plate, and the dramatic narrowband perfect absorption peak with the FWHM  of 300 nm hence is achieved. The spectral position of the absorption peak can be dynamically tuned by a small change in the chemical potential of graphene, in addition to varying geometrical parameters of the absorber. Meanwhile, this device exhibits good absorption stability over a wide angle range of incidence around ± 60° at least. Such absorber will benefit the fabrication of mid-infrared nano-photonic devices for optical filtering and storage. Over the past decades, an extensive amount of research has been carried out on the metamaterials19111518192022Recently, graphene24272830313234Currently, a great diversity of graphene-based metamaterials3738x-y cross-section of this device with the corresponding geometrical description is shown in the inset. The double-layer graphene ribbons with identical width of W are separated by the silica layer with the thickness of d. The period of the ribbons array is P. The cavity between the metallic ground plate and the lower graphene layer is filled also by the silica layer with thickness of D. The thickness of the metallic ground plate is H. When the plane wave with the polarization along the x axis irradiates normally this structure, the second-order dipole resonance should form on the single graphene ribbond = 50 nm, the anti-symmetric plasmonic dipole-dipole coupling resonanceT = 0), as the thickness of the metallic ground plate with H = 800 nm is much larger than the penetration depth of electromagnetic waves. When the double-layer graphene ribbons are considered as a whole and called the anti-symmetric dipole-dipole coupling resonator, the proposed device behaves as such construction of the single port coupled with a resonator. The versatile CMT4647First, the structure of the proposed absorber composed of the periodic double-layer graphene ribbon array placed near a metallic ground plate coated by a dielectric layer is depicted schematically in the S+, and S−, respectively. The resonant frequency of the anti-symmetric dipole-dipole coupling mode formed between double-layer graphene ribbons is ω0. The dissipative and radiative losses occurring in the anti-symmetric coupling resonator are represented by τa−1 and τr−1, respectively. The coupling strength between the incident wave and the anti-symmetric resonator is characterized by τc−1. When the incident wave with the frequency ω is launched into this system, the time-evolution-normalized amplitude of the anti-symmetric dipole-dipole coupling resonator is expressed asAs shown in the inset of Thanks to the power conservation and time reversal symmetry, the relationship between the amplitudes of the normalized incoming and outgoing waves transmitted through the only port is described asr0 is the reflectance of this system without the double-layer graphene ribbon array. Because the metallic ground plate behaves as a mirror, all incident waves will be reflected directly at the upper surface of the metallic plate. Therefore, we can obtain r0 = −1. j stands for the imaginary unit. Isolating the S−/S+ and going to the frequency domain ejωt+ , the reflectance of the device can be obtained and expressed asHere, the ω = ω0 for the reflection spectrum. At the ω = ω0, the effective impedance of this system is z0 = 1), the radiative losses can be neglected, corresponding to τr−1 ≈ 0. Hence, only the condition of τa−1 = τc−1 should be satisfied. At the ω = ω0, the impedance matching is realized and r = 0. Thus, the absorption is A = 1 − T − |r|2 = 1. In other word, the perfect absorption response will be achieved at the ω = ω0.The impedance of this system can be defined as The reflection of this system isThe absorption of this structure can be given byτa−1 = τc−1 = 1*1012 Hz are considered for obtaining a useful impedance matching. The resonant wavelength λ0 = 2πc/ω0 is assumed as 11.16 μm, here c is the velocity of light in vacuum. Obviously, the perfect absorption feature occurs. In order to confirm the CMT-predicted perfect absorption response and further investigate comprehensively the proposed absorber, the numerical simulation is performed by the Lumerical FDTD Solutions.The CMT predicted absorption spectrum is shown in y direction). The surface conductivity (σg) of the graphene is governed by the Kubo formula49τ, temperature T, chemical potential μc, and incident wavelength λ (frequency ω). At the room temperature and for the highly doped graphene in the simulated mid-infrared spectral range, the chemical potential is always above half of the photon energy. The Kubo equation hence is reduced toεeq of graphene is thickness-dependentμc = 0.4 eV, and the momentum relaxation time is chosen as τ = 0.5 ps. The metal is chosen as the silver with the dispersive permittivity characterized by the Drude model5354ε∞ = 3.7 is the dielectric constant at infinite angular frequency; ωp = 9.1 eV is the bulk plasma frequency representing the natural frequency of the oscillations of free conduction electrons; γ = 0.018 eV stands for the damping frequency of the oscillations. In the considered mid-infrared region, the silica is assumed to be a non-dispersive dielectric with the index of 1.45. The unit cell with P = 300 nm, W = 150 nm, and D = 600 nm is performed. Periodic boundary conditions are applied along the y directions and perfectly matched layers are employed in the x direction. A plane wave with the electric field parallel to the x axis illuminates normally the periodic structure. We use the non-uniform mesh, and the minimum mesh size inside the graphene layer equals 0.1 nm and gradually increases outside the graphene sheet, for saving storage space and computing time. 2D-FDTD simulated results are presented in In the numerical calculations, the graphene ribbon is modeled as an ultra-thin film with a thickness of Δ  distributions of the entire unit cell at wavelengths of λ0 = 11.16 μm and λ = 14.00 μm, are shown in λ0 = 11.16 μm, the electrical resonance yields at the anti-symmetric coupling plasmonic resonator formed by the double-layer graphene ribbons. The effective impedance of the vertically coupling graphene ribbons matches to that of the free space, and the reflection thus is suppressed. The transmission from this system is also inhibited due to the ultra-thick metallic ground plate. All incident electromagnetic energy is efficiently confined in the silica interlayer between the graphene ribbons and the metallic ground plate. Therefore, the perfect absorption feature is achieved by the simple graphene-based structure. On the contrary, the electrical resonance of the double-layer graphene ribbons and the effective impedance matching condition cannot be formed at the other nor-resonant wavelengths. The most incident power is reflected and cannot be confined by this device, as shown in λ = 14.00 μm.In the d = 50 nm and the duty ratio of W/P = 0.5 are fixed. Other structural and material parameters are unchanged. The structure with the simultaneous change in the width W of double-layer ribbons is simulated and results are presented in W = 150, 180, 210, and 240 nm are offered. The obvious width-dependent tunability is achieved. In addition, the real part of the wave vector of the anti-symmetric dipole-dipole coupling resonance increases with the vertical coupling distance between ribbons increased increases. Simulated absorption spectra with different coupling distances are displayed in Now that the spectral position of the perfect absorption peak corresponds to the resonant wavelength of the anti-symmetric coupling dipole-dipole resonator formed on the double-layer ribbons, the change in the resonant wavelength of the anti-symmetric coupling resonator can be utilized to tune the spectral position of the absorption peak. For anti-symmetric coupling dipole-dipole resonator, the changes in the geometrical parameters of double-layer graphene ribbons should have a direct effect on its resonant wavelength. Thus, the changes in the geometrical parameters of graphene ribbons can be used first for tune the spectral position of the absorption peak D are presented in D has little effect on the central wavelength of the absorption peak, whereas influences greatly the absorption and FWHM of the resonant peak. In other word, the absorption spectrum can be modulated by changing the thickness D, without changing the spectral position of the absorption peak. This phenomenon offered by the D of the silica spaceD increases from 50 to 600 nm, the perfect impedance matching to that of the free space is formed gradually. With the thickness D increase further, the perfect impedance matching is broken. Hence, the perfect absorber is achieved first and then is hampered with the increase of the thickness D. On the other hand, the anti-symmetric dipole-dipole coupling plasmonic resonance in double-layer graphene ribbons is localized around the graphene ribbons with deep sub-wavelength nano-scales, which are less than 50 nm as shown in the inset of D increases from 50 to 1000 nm.In addition, for the absorption peak, the most incident electromagnetic power is confined in the space between the lower graphene ribbon and the metallic ground plate. Hence, the height of the space should have a direct effect on the performance of this absorber. Numerical absorption spectra under different distances μc = 0.35, 0.4, 0.45, and 0.5 eV, respectively, are shown in Besides the geometrical tunability, the graphene-based absorber is particular to own the novel gate-voltage-dependent feature that the chemical potential of graphene can be dynamically tuned by applying external gate voltages, as shown in It should be pointed out that the electromagnetic waves irradiate usually this absorber with an oblique incident angle in the practical applications. Thus, it is necessary to investigate the robustness of the proposed perfect absorber under the oblique incidence. Other simulation parameters are similar to that in To sum up, the periodic double-layer graphene ribbon arrays placed on a metallic ground plate coated by a dielectric space are proposed and investigated both numerically and theoretically. The well-known CMT is used for analyzing the structural property and predicting the perfect absorption feature. FDTD numerical simulation is utilized to give the underlying physics. Simulated results exhibit that the localized anti-symmetric dipole-dipole coupling modes occurs at double-layer graphene ribbons and acts as the electrical resonance to suppress the reflection, thanks to the impedance matching. The ultra-thick metallic ground plate is used for restricting the transmission from this system. The incident electromagnetic energy is efficiently confined in the interlayer between graphene ribbons and the metallic substrate, and the novel perfect absorber hence is achieved. The spectral position of the absorption peak can be dynamically tuned by a small change in the chemical potential, in addition to varying geometrical parameters of the absorber. Meanwhile, this device exhibits good absorption stability over a wide angle range of incidence around ± 60° at least. Such simple plasmonic perfect absorber may benefit the fabrication of nanophotonic devices for optical filtering and storage in the mid-infrared region.y directions and perfectly matched layers are employed in the x direction. A plane wave with the electric field parallel to the x axis illuminates normally the periodic structure. The non-uniform mesh is adopted, and the minimum mesh size inside the graphene layer equals 0.1 nm and gradually increases outside the graphene sheet, for saving storage space and computing time.The 2D finite-difference time-domain (FDTD) method is used for the numerical simulation and the commercial software of Lumerical FDTD Solutions is performed. Periodic boundary conditions are applied along the How to cite this article: Li, H. et al. Tunable graphene-based mid-infrared plasmonic wide-angle narrowband perfect absorber. Sci. Rep.6, 36651; doi: 10.1038/srep36651 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Testicular germ cell tumors (TGCTs) are nearly universally curable malignancies. Nevertheless, standard cisplatin-based chemotherapy is not curative in a small subgroup of patients. Previously, we showed that PD-L1 overexpression is associated with worse prognosis in TGCTs, while tumor infiltrating lymphocytes (TILs) are prognostic in different types of cancer. This study aimed to evaluate the prognostic value of PD-1 and PD-L1 expressing TILs in TGCTs.PD-L1 positive TILs were found significantly more often in seminomas (95.9% of patients) and embryonal carcinomas (91.0%) compared to yolk sac tumors (60.0%), choriocarcinomas (54.5%) or teratomas (35.7%) (All p < 0.05). TGCTs patients with high infiltration of PD-L1 positive TILs (HS ≥ 160) had significantly better progression-free survival  and overall survival  opposite to patients with lower expression of PD-L1 (HS < 150). PD-1 expressing TILs were not prognostic in TGCTs.Surgical specimens from 240 patients with primary TGCTs were included into this translational study. The PD-1 and PD-L1 expression on tumor and TILs were detected by immunohistochemistry using anti-PD-1 and anti-PD-L1 monoclonal antibody. Scoring was performed semiquantitatively by weighted histoscore (HS) method.The prognostic value of PD-L1 expressing TILs in TGCTs was demonstrated for the first time. Testicular germ cell tumors (TGCTs) are nearly universally curable malignancies, most commonly found in young men . CisplatMalignant tumor biology and its’ associations with immune mechanisms have been recently a subject of intensified research. It occurs that immune check-point inhibition including PD-1, PD-L1 and/or CTLA4 is a highly effective modality in several types of malignancies –10. Tumo+ T cells, CD8+ T cells, natural killer cells, B cells and monocytes : AB174838) diluted 1:100 in Dako REAL antibody diluent  and immunostained using anti-mouse/anti-rabbit immuno-peroxidase polymer  for 30 minutes at room temperature, according to the manufacturer's instructions. Color reaction was developed with diaminobenzidine substrate-chromogen solution  for 5 minutes. Finally, the slides were counterstained with haematoxylin, mounted and reacted for 5 minutes with diaminobenzidine substrate-chromogen solution  for visualization. PD-1 and PD-L1 positivity of lymphocytes in the tonsil was used as a positive control, same tissue with omitting of the primary antibody served as negative control.Tissue samples were evaluated under a light microscope , the microphotographs were acquired using the camera  and software DSLR Remote .Tumor cores were independently assessed by two observers (ZC and PB) who were blinded to clinicopathological data. In cases of disagreement, the result was reached by consensus. TILs were identified in hematoxylin-eosin staining according to typical morphology Figure . TILs wiPatients’ characteristics were tabulated. A distri-bution of PD-1 and PD-L1 HS in TILs was significantly different from the normal distribution (Shapiro–Wilk test), therefore we used non-parametric tests for analyses. Analyses of differences in distributions of PD-1 and PD-L1 expression in TILs between the two groups of patients were performed using the Mann–Whitney U test, while Fisher's exact test or the χ2 test when appropriate were used, when PD-1 and PD-L1 expression was categorized as ‘low’ or ‘high’ according to the aforementioned criteria.Median follow-up period was calculated as a median observation time among all patients and among those still alive at the time of their last follow-up. Progression-free survival (PFS) was calculated from the date of orchiectomy or the date of tumor biopsy to the date of progression or death or the date of last adequate follow-up. Overall survival (OS) was calculated from the date of orchiectomy or date of tumor biopsy to the date of death or last follow-up. PFS and OS were estimated using the Kaplan–Meier product limit method and compared by the log-rank test. A multivariable Cox proportional hazards model for PFS and OS was used to assess differences in outcome on the basis of PD1 and PD-L1 expression in tumor cells and TILs of primary tumor and prognosis according to IGCCCG  . All rep


Unlike sensitivity, response- and recovery-time demonstrate directly proportional dependence with operating temperature. Power dependent studies establish linear relation between power-density and sensitivity, and a safe limit beyond which sample heating prolongs the recovery time. Wavelength-dependent studies shows that proposed sensor can efficiently operate from visible to near NIR region. To the best of our knowledge such rGO based optical sensor performance at low temperature had not been reported earlier.We report a facile and cost-effective approach to develop self-standing reduced Graphene Oxide (rGO) film based optical sensor and its low-temperature performance analysis where midgap defect states play a key role in tuning the crucial sensor parameters. Graphite oxide (GO) is produced by modified Hummers’ method and reduced thermally at 250 °C for 1 h in Argon atmosphere to obtain rGO. Self-standing rGO film is prepared via vacuum filtration. The developed film is characterized by HRTEM, FESEM, Raman, and XRD techniques. The developed sensor exhibits highest sensitivity towards 635 nm illumination wavelength, irrespective of the operating temperature. For a given excitation wavelength, photoresponse study at low temperature (123K–303K) reveals inverse relationship between sensitivity and operating temperature. Highest sensitivity of 49.2% is obtained at 123 K for 635 nm laser at power density of 1.4 mW/mm In this regard, apart from the choice of material, dimensionality also significantly affects the sensor performance. Recent advancements in the frontiers of nanotechnology have opened new avenue and pathways for researchers to look for novel materials for a variety of applications. Graphene has turned out to be one of the most celebrated inventions in post silicon era because of its extraordinary and interesting properties: small thickness (of the order of one atomic layer), large surface to volume ratio, very low mass, high mobility of charge carriers, and decent absorption coefficient, etc.8.Photodetectors having superior detection parameters such as sensitivity, speed of response, internal as well as external quantum efficiency, detection bandwidth and flexibility are in great demand for their widespread applications in sensing, imaging, etc.π-π* band structure. At Dirac point  the conduction and valence bands are symmetrical. The superior electrical and optical characteristics in graphene are because of linear dispersion of Dirac massless electrons moving with a fraction of the speed of light. In pure graphene, fermi energy lies at charge neutrality point, so its electronic properties near k-point (Dirac point), the conductance is expected to be a minimum. Under external stimulus, the entire band structure can be shifted with respect to the fermi level and pave the way to induce enhanced conductivity via: (a) increasing the magnitude of gate voltage (in FET)11, (b) excess electron-hole pair generation by irradiating light (in photoconductor)12, and (c) enhanced electron-phonon scattering  on either side of the Dirac point11.Graphene is a semi-metallic material because of its special 14. On the contrary, its poor photo-responsivity14 and extremely short carrier lifetime (of the order of picoseconds)3, which originates from its intrinsic zero-band gap energy, does not allow graphene to be the best candidate for photodetection. One way to solve this issue is bandgap opening14. Defect engineering is a versatile option through which mid-gap states can be created within the band structure of graphene to resolve two prominent issues simultaneously: (a) to slow down the carrier recombination time by charge trapping, and (b) increase in bandgap also increases the absorption efficiency compare to pristine graphene14. An easy way to achieve this is to transform graphene oxide (GO) to reduced graphene oxide (rGO). The band gap in GO is ~2.2 eV and for rGO, bandgap can vary from ~1.00 to 1.69 eV depending on the degree of reduction19. On reduction, some of the oxygen groups are removed and bandgap can therefore be adjusted further by managing the oxygen present in rGO. Thus, reduced graphene oxide (rGO) behaves like semi-metal or semiconductor and its electrical conductivity may be tuned by controlling its oxygen content16.A good photodetector should detect light of a wide spectral range. Graphene’s capacity of absorbing ~2.3% of incident light and its high mobility renders the realization of broadband photodetector with ultrafast detection (>500 GHz)17. Very recently, pristine graphene based infrared photodetectors are demonstrated with an optical modulation of up to 40 GHz, however a low responsivity (~6.1 mA/W), caused by rapid photocarrier dynamics, is compromised18. In 2013, Haixin et al. reported photoresponse of rGO based phototransistor operated in infrared region by controlling the defects and atomic structure; remarkable infrared photoresponse of ~0.7 A/W (at 895 nm) and external quantum efficiencies (EQE) of 97% is reported19. D. Sun et al. reported ballistic photocurrent generation in a graphene based photodetector20. Notwithstanding the gapless nature of graphene, strong photocurrent response is reported at metal/graphene interface having internal quantum efficiency ~15–30%21. Chang-Hua Liu et al. designed a wide-broadband photodetector using graphene bi-layer heterostructure22. In 2017, Jing Jing et al. have reported ultra-high responsivity of upto 105 A/W at room temperature in silicon-graphene conductive photodetector23. All these disparate responses are found due to varying types of graphene synthesis routes, methodology, device engineering and fabrication.In 2014, Haifeng Liang has reported mid-IR photoresponse of rGO with a responsivity of 1 A/W24. Other disadvantage is its less absorption area, which in turn limits the responsivity of sensor. Hence, to enhance the photo-response, we need to focus on three prominent factors – (a) design a sensor that provides sufficient absorption area, (b) convert the precursor graphite powder to rGO to enable maximum light absorption for generation of e-h pairs in excess, and (c) drift the generated carriers towards electrodes to avoid band to band recombination, otherwise ultrafast (~ps) recombination of e-h pairs will fail the prospects of the sensor due to its zero-bandgap nature. On the flip side, we sacrifice the speed of the device because defects slow down the mobility of charge carriers and narrow down the detection bandwidth3. Researchers have been trying to trade-off this dilemma by considering several aspects including material synthesis, device design and fabrication strategies. Even efforts were made to develop rGO mesh/network structure by spray deposition as well as synthesizing composite with polymers, glues, epoxies, etc.Sensor design by placing a monolayer graphene between the electrodes in lateral configuration is difficult and an expensive task, since it requires high-end nanoscale manipulation techniques26. The processed film is a mesh of well dispersed graphene sheets with an average size of ~2 μm. The device performance has been studied against various sensor parameters such as excitation wavelength (visible to NIR), illumination power density and low temperature. Unlike sensitivity, response- and recovery-time demonstrate directly proportional dependence with operating temperature. Highest sensitivity of 49.2% is achieved at 123 K for 635 nm wavelength at 1.4 mW/mm2 laser power density, indicating the potential of the sensor to operate even in cryogenic conditions. Power dependent photoresponse indicate linear variation of sensitivity with power density upto certain extent beyond which bolometeric effect comes into action and extends the sensor recovery time. The sensor exhibits high sensitivity for wide range of illumination from Vis to NIR signifying its potential device applications for wide bandgap photodetectors.In this paper, we report the temperature dependent photo-sensing response of rGO based photo-detector. The free-standing rGO film is developed by vacuum filtration technique27: A round bottom flask is kept in an ice bath and filled with 120.0 mL H2SO4 (95%). Graphite powder (5.0 g) and NaNO3 (2.5 g) are added to the flask under vigorous stirring. Followed by this step, 15.0 g KMnO4 is mixed under continued stirring at a temperature of less than 10 °C. After mixing KMnO4, ice bath is taken off and solution is stirred at 30 °C for a day. The mixture progressively grew thick (paste) and turned light brown in color. Following this, 150.0 mL of deionized water is gradually added in this mixture under constant stirring. The diluted suspension is stirred for 2 h at 98 °C. Subsequently, the temperature is decreased to 60 °C, and 50.0 mL H2O2 (30%) is further added in the mixture in order to remove any leftover MnO−4. Lastly, the resultant mixture is filtered and rinsed with deionized water to obtain neutral pH. The extracted mixture is then desiccated at 80 °C and graphite oxide is obtained as powder which is exfoliated to GO sheets using a ultrasonication  followed by centrifugation (3000 rpm) to separate unexfoliated graphite oxide.Graphene oxide (GO) is produced from graphite powder using modified Hummers’ techniqueUltrasonication time is varied  in order to obtain monolayer GO sheets. This was followed by vacuum filtration by using cellulose nitrate filter membranes . Further, the film is desiccated at 60 °C in a hot air oven for 24 h and peeled off from the filter as self-standing GO film. Finally, this film is thermally reduced at different temperatures  in inert atmosphere for different annealing times  to optimize the film quality to achieve best performance.sp3 matrix  network of as produced GO29. The structural disorder-induced localized states might be present in the band tail of π–π* gap or lie deep inside this gap. Accordingly, optical transitions involving these disorder-induced localized states may cause wide absorption or emission bands30.The understanding of lattice as well as electronic band structure of graphene, GO and rGO is necessary to understand how the transformation of GO to rGO leads to high photoexcitation response. The atomic structure of GO is purported as a graphene basal plane having inconsistently dispersed oxygen-containing functional groups (hydroxy and epoxy units) resulting in rix Fig.  which ma29. As a result, certain carbon lattices from the initial distorted sp2 domains may create additional graphitic domains of sp2 clusters29. These small sp2 clusters, create isolated molecular states, finally percolate to facilitate the charge carrier transport by hopping28. The reduction process leads to increased carrier mobility, higher absorption, a tunable bandgap where photoresponsivity can be controlled by controlling the defects and oxygen groups29.The quantity of disorder-induced states declines after reduction owing to deoxygenation. This structural modification is attributed to the reduction of oxygenous functional groups and gradual recovery of the conjugations between carbon atoms during reduction processSurface morphology of the self-standing film prepared by vacuum filtration is thoroughly examined by Field Emission-Scanning Electron Microscope . The SEM image of film is uniform with little wrinkles as evident in the SEM micrographs shown in Supplementary Information Figure  provides2 30 S Twin) images of GO sheets that are highly transparent with some overlaps and wrinkles. From figure it is clearly visible that as ultrasonication time is increased , the transparency of the sheets has enhanced significantly and GO sheets can be exfoliated to single layer by increasing the ultrasonication time. We have produced GO that has single as well as few layer flakes/ribbons with an average size of ~2 µm.Figure −1 and 1580.4 cm−1, arising due to vibrations of sp2 carbons. The G- and D-peak intensities are important to measure the graphitic character and the degree of disorders respectively. In GO, large band width is indicative of large structural disorders. The 2D peak occurs at 2706 cm−1 in GO and rGO and intensity is characteristically weak as compared to pristine graphene. Figure Raman characterization is an extensively employed technique to get the structural information of the material. Characteristic peaks in the Raman spectra of graphene are designated as D (disorder), G (graphitic), and 2D. The first order D and G peaks appear at ~1350 cmθ = 10°). The rGO sample shows a new strong peak at 2θ = 25.1° and it is attributable to the removal of oxygen containing functional groups, resulting in reduced d-spacing compare to GO.Figure π-π* transition of aromatic C-C bonds. Absorption peak for rGO is red shifted to 266 nm, and this shift is considered as a measure for the reduction of GO31. Films obtained from vacuum filtration method is thermally reduced at various temperature  in inert atmosphere for different times . Figure Figure 32. The fabricated rGO film in this device configuration works as a photosensitive sensor and its conductance increases upon light illumination.Optical sensors were developed from the self-standing rGO film by cutting 1 cm × 1 cm pieces and depositing silver electrodes on both ends. Figure exc ~ 635 nm, 785 nm, and 1064 nm of tunable powers. The distance between laser source and sample was kept constant at 10 mm with a spot size of approximately 6 mm comfortably confined within the sensing area. The laser power was recorded using a Newport 843 R power meter.The sensor was placed in the sample chamber , and the electrical signal is measured with Keithley SCS 4200 system. The light from diode laser source is irradiated on the sensor from top side, as shown in Fig. 35. We can exclude the photogenerated thermoelectric effect for photocurrent, because a homogeneous temperature field can be achieved in the device by focusing light to cover the entire device and the photothermal currents flowing in two different directions are cancelled out on contacts36. This is evident in our sensor recovery plot as evident in the studies carried out in the section Temperature dependent Sensing Response. Any heating effect makes a slow exponential decay38 and this is absent in our case. Moreover, as evident from I-V characterization shown in Fig. sc), therefore, the photovoltaic effect may be ruled out. These results strongly suggest that the photoconductive effect is the dominant mechanism of photocurrent generation in the rGO based photodetector33.As recognized, transformation of GO to rGO makes rGO flakes which is nothing but stacking of graphene-like layers. For layer based photodetectors, even applicable to rGO, three types of main photocurrent generation mechanisms are proposed: photoconductive effect, photovoltaic effect, and photothermoelectric effect− and h) recombine together39. While drifting within the channel space, photogenerated carriers may get confined to trap/defect states present within the bandgap as well as at the rGO/electrode interface, and subsequently de-trap by incident photons or thermal vibrational energies. Therefore, under light illumination, confined carriers tend to return to their respective bands instead of recombining; this is because- (a) the incident photons have much higher activation energy than the trap barrier potential, and (b) photon flux is sufficient to reduce the effective recombination rate of carriers40.The general operational principle of a solid-state photodetector involves: (a) generation of carriers by absorbing incident photons within the semiconducting layer, (b) transport and multiplication (if available) of these carriers, and lastly (c) driving out these carriers into the external circuit to circulate until the photogenerated carriers 43 and the other is the trap involving Auger- recombination. (RAuger)47. In the limit of high carrier density, these can be simply expressed as35:αe(h) represents the capture cross sections to trap electrons (holes), and Nt is the areal density of traps. Under illumination, the exchange of electrons mainly takes place between bands and trap/defect states; therefore, any variation in number of electrons either in the conduction band or in trap/defect states must include all possible processes that take part such as -Change in the number of conduction band electrons occurring due to photogeneration, carrier multiplication (carrier impact ionization), recombination and relaxation to the defect states, andChange in the quantity of electrons on the defect states occurring due to excitation to the conduction band, relaxation from conduction band, and recombination with holes in the valence band.At any temperature, the principle of detailed balance between generation and recombination may be worked out considering the interband as well as Auger recombination processes in case of graphene and related materials. A detailed analysis is available in the literature and readers may refer to the references thereinα) of the trap/defect states as described by Yongzee Zhang et al.3 isη) of the photoexcited and secondary-generated electrons trapped by defect states is written as3,χ is the number of electrons per absorbed photon due to electron and hole impact ionization, and β represents photogeneration rate.Considering these facts, the electron capture rate (R is the electron recombination rate.From Eqs  and 4),,4),5\docα and R are temperature dependent. In case of graphene, R ~ 1 ps43 and assumed to remain unchanged due to negligible change in its order at higher temperature. Therefore, α, the electron capture cross-section rate by the defect states, will be the ultimate decisive factor to control η; In a nut shell, photoresponsivity (Rλ) as well as the external quantum efficiency (EQE) (refer to Supplementary information file) of the sensor device is the outcome of both the factors α and η for low and high temperature of operations.In Eq. , both th44 as evident in the studies carried out in the section Temperature dependent Sensing Response. Photogenerated electrons can be trapped in the defect states and remain there at temperature not sufficient to overcome the capture probability of the defect states. Owing to charge conservation in the carrier conduction channel, multiple hole-circulation occurs after a single photon generates an e-h pair, and the holes do not recombine with electrons until the dilution of the said capture probability46.There are many defects or trapping states in multilayered rGO flakes that influence free charge carrier density in the conduction band. It is supported by the linear dependence of photoconductivity on light power densityDecrease in electron capture probability with the defect states leading to release/generation of free carriers to the conduction band. This corresponds to the transition of electrons from defect energy state (lying below conduction band) to conduction band.Electron-hole pair non-radiative recombination through electron transition from conduction- to valence band leading to loss of electron and hole together in their respective bands.47.The factors that delay the recombination process as well as increase the electron transit time  of the order 10m+n, where m and n are the scaling exponent factor and their values are >>139.Factors (a) i.e. the electron capture probability is concerned with the defect energy states confined in the forbidden gap, and it is inversely proportional to the temperature of operationbias) across the sensor device to drift the carriers and this is how the photoconductive detector works. In addition, this recombination loss probability is further reduced due to the random scattering factors (c-g). Higher the temperature, higher the scattering, and lesser is the recombination probability. As the scattering factors increase, the carrier transit time , leading to a decrease in collection of net charge carriers (Q) at the terminal electrode48; this will reduce the terminal current I  of the sensor device should increase with increase in temperature due to the dominance of carrier generation factor over the delay in transit time. Temperature dependent experimental results endorse these facts and suitable analysis is given in the next sections.The second factor (b) i.e. the ‘e-h pair non-radiative recombination’ should theoretically reduce the number of carriers but this is prevented to a very large extent by applying the optimized bias voltage  while intensity of incident light was kept constant. Figure 3 carbon bonding is dominant  mechanism57. Each defect site is a charge trapping center and therefore, electron mobility or electron transit time between terminal electrodes heavily depend on the density of defects, which, in turn depends on thermal reduction time from GO to rGO transformation. The experimental results are in close agreement of these arguments are shown in subsequent sections. Figure Figure exc) is depicted in Fig. exc  based self-standing film is produced via modified Hummers’ technique and subsequently annealed at different temperatures for varying time durations. Raman and XRD analysis verified the degree of reduction during transformation from GO to rGO. Optical sensor is developed from the self-standing rGO film. Sensor is exposed to visible to NIR excitations and highest response is observed under 635 nm laser illumination at room temperature. Temperature dependent studies suggest an inverse relation between sensitivity and operating temperature. Highest sensitivity of 49.2% is obtained at 123 K for 635 nm laser with power density of 1.4 mW/mmFigure S(I), Figure S2