Vitis sp. cv. Koshu, inhibited mycelial growth of Botrytis cinerea, Colletotrichum gloeosporioides, and Phytophthora infestans. The KOF112-inhibited mycelial tips were swollen or ruptured, suggesting that KOF112 produces antifungal substances. Analysis of the 16S rDNA sequence revealed that KOF112 is a strain of Bacillus velezensis. Comparative genome analysis indicated significant differences in the synthesis of non-ribosomal synthesized antimicrobial peptides and polyketides between KOF112 and the antagonistic B. velezensis FZB42. KOF112 showed biocontrol activities against gray mold caused by B. cinerea, anthracnose by C. gloeosporioides, and downy mildew by Plasmopara viticola. In the KOF112–P. viticola interaction, KOF112 inhibited zoospore release from P. viticola zoosporangia but not zoospore germination. In addition, KOF112 drastically upregulated the expression of genes encoding class IV chitinase and β-1,3-glucanase in grape leaves, suggesting that KOF112 also works as a biotic elicitor in grapevine. Because it is considered that endophytic KOF112 can colonize well in and/or on grapevine, KOF112 may contribute to pest-management strategies in viticulture and potentially reduce the frequency of chemical fungicide application.As the use of chemical fungicides has raised environmental concerns, biological control agents have attracted interest as an alternative to chemical fungicides for plant-disease control. In this study, we attempted to explore biological control agents for three fungal phytopathogens causing downy mildew, gray mold, and ripe rot in grapevines, which are derived from shoot xylem of grapevines. KOF112, which was isolated from the Japanese indigenous wine grape  Vitis sp. cv. Koshu, a hybrid of Vitis vinifera L. and V. davidii Foex, is an indigenous wine grape in Japan . B.. B.B. ciere used ,48. P. iBacillus isolates deposited in the NCBI database. The nucleotide sequences were subjected to the NJ method using Molecular Evolutionary Genetics Analysis software, MEGA10 .Genomic DNA was extracted from the one-day culture of KOF112 in SCD medium using a DNeasy PowerSoil Kit  in accordance with the manufacturer’s instructions. PCR conditions for amplifying partial 16S rDNA were as follows: after incubation at 94 °C for 5 min, PCR amplification was performed for 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with a final extension step at 72 °C for 7 min. The nucleotide sequences of the primer set for amplifying partial 16S rDNA from bacterial genome were as follows: 795F (5′-GGATTAGATACCCTGGTA-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′). The nucleotide sequences of the amplicons were analyzed using the dye terminator method and subjected to the Basic Local Alignment Search Tool . Phylogenetic analysis was performed using the partial 16S rDNA of KOF112 and DNA extraction from KOF112 was performed using a DNeasy PowerSoil Kit (Qiagen). Genome sequencing, assembly, and annotation were performed as described previously . BrieflyB. velezensis FZB42  and B. amyloliquefaciens DSM7 (accession no. FN597644) were used as representative antagonistic Bacillus species for comparative genome analysis. Comparative genome analysis was performed using the CGView Server [Genome sequences of w Server .8, 1 × 107, 1 × 106, and 1 × 105 cfu/mL with SCD medium and sterile water . Ten microliters of KOF112 solution was dropped onto four locations on the abaxial surface of the leaf disk. Ten percent SCD medium and Agrobacterium sp. isolate CHB3  were used as control. The leaf disks were dried at room temperature in a flow cabinet for 3 h.An in vivo bioassay using grape leaf disks was performed as described previously, with minor modifications . BrieflyP. viticola zoosporangia was performed as described previously with minor modifications [P. viticola was maintained on leaves of potted Koshu seedlings in a growth chamber under light irradiation (11.8 Wm−2/16 h/d) at 21 °C. Fresh zoosporangia of P. viticola were washed off with sterile water from the symptoms on the Koshu leaves. Ten microliters of the zoosporangium suspension (1 × 104 zoosporangia/mL) was dropped onto the same locations as the pretreated KOF112 solutions on the leaf disks. The Petri dishes containing the leaf disks were placed in a plastic box containing moistened paper towel. The box was incubated in the dark for 24 h and then in an incubator . Downy mildew symptoms on each disk were assessed 7 days post P. viticola inoculation. Disease severity was scored by evaluating the symptom on each disk as described previously [Inoculation of ications . Brieflyeviously : 0, no sCucumis sativus cv. Sharp-1) seedlings and sprayed with 500 μL of KOF112 solution  with a hand sprayer. As the control experiment, sterile water, 10% SCD medium, or Agrobacterium sp. isolate CHB3  was sprayed. After air drying the cotyledons, the center of each cotyledon was punctured with a sterile needle. Mycelial disks (6.5 mm diameter) of B. cinerea laboratory strain were excised from the leading edge of the colonies on PDA plates, and a disk was placed on the wound of each cotyledon. After incubation in a moisture chamber at 25 °C for 6 d in an incubator (11.8 Wm−2/16 h/d), disease severity was scored by evaluating the symptom of each cotyledon as described previously [Three true leaves (approximately 4 cm in size) were detached from cucumber (Fragaria ananassa cv. Hokowase) were detached from seedlings cultivated in a growth chamber. Three detached leaves were sprayed with 500 μL of KOF112 solution (1 × 108 cfu/mL) with a hand sprayer. As the control experiment, sterile water, 10% SCD medium, or Agrobacterium sp. isolate CHB3  was sprayed. After air drying the leaves, the center of each leaf was punctured with a sterile needle. Mycelial disks (6.5 mm diameter) of C. gloeosporioides laboratory strain were excised from the leading edge of the colonies on PDA plates, and a disk was placed on the wound of each leaf. After incubation in a moisture chamber at 25 °C for 8 d in the dark, disease severity was scored by evaluating the symptom of each leaf as described previously [Strawberry leaves  was prepared as mentioned above. Nine hundred microliters of the zoosporangium suspension was incubated with 100 μL of KOF112 solution , 10% SCD medium, or Agrobacterium sp. isolate CHB3  in a microtube at 22 °C for 1 h under light irradiation (11.8 Wm−2), and then for 23 h in the dark. Zoospore release from zoosporangia was observed under a light microscope . Zoospore release rate was calculated using the following formula:A zoosporangium suspension of P. viticola (1 × 104 zoosporangia/mL) was prepared as mentioned above. Five hundred microliters of the zoosporangium suspension was incubated in a microtube at 30 °C for 4 h under light irradiation (11.8 Wm−2). One hundred microliters of KOF112 solution , 10% SCD, or Agrobacterium sp. isolate CHB3  was added into the microtube, and then incubation was carried out at 30 °C for 12 h under light irradiation (11.8 Wm−2), followed by incubation at 30 °C for 8 h in the dark. The suspension was stained with 0.05% aniline blue in 0.0067M K2HPO4 (pH 9–9.5) at room temperature for 20 min. Zoospore germination was observed using a fluorescence microscope (Olympus BX51). Zoospore germination rate was calculated using the following formula:Zoospore germination was counted as described previously, with minor modifications . Briefly8 cfu/mL, containing 10% SCD) was dropped onto four locations on the abaxial surface of the leaf disk. Ten percent SCD medium or Agrobacterium sp. isolate CHB3  was used as control. After incubation at 22 °C for 24 h and 48 h in an incubator (11.8 Wm−2/16 h/d), the disks were homogenized in a mortar containing liquid nitrogen using a pestle. Total RNA isolation from the pulverized samples was performed using NucleoSpin RNA Plant , and purification was carried out using Fruit-mate for RNA Purification (Takara). First-strand cDNA was synthesized from the total RNA using PrimeScript RT Master Mix  (Takara). Real-time RT-PCR was performed using an SYBR Premix Ex Taq II  (Takara) with a Thermal Cycler Dice Real Time System (Takara). PCR conditions were as follows: incubation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and at 60 °C for 45 s. The primers used for amplification were as follows: ubiquitin primers ; class IV chitinase primers , and β-1,3-glucanase primers . Ubiquitin primers were used as the reference gene to normalize each gene expression because ubiquitin gene expression was stable in the grapevine leaves [To determine whether KOF112 induces plant defense response in grapevines, transcriptional alteration of genes encoding two PR proteins, class IV chitinase and β-1,3-glucanase, was evaluated in KOF112-treated grapevine leaves. Three leaf disks each having a diameter of 13 mm were cut out from Koshu leaves using a cork polisher and placed upside down on moistened filter paper in square Petri dishes (140 mm × 100 mm). Ten microliters of KOF112 solution .Data are presented as means ± standard deviations of biological replicates. Statistical analysis was performed by the Student’s


Citrus aurantiifolia) is a plant belonging to the family of Rutaceae and to the genus Citrus. The fruit is widely used in the United States, Mexico, Southeast Asia, Latin America, but is increasingly widespread all over the world. It is used as a fresh fruit, in the preparation of foods, sweets and drinks and its oils are used in the cosmetic and pharmaceutical industry. The main adverse reactions to lime seem to be represented by contact dermatitis, allergic and phototoxic type. In the context of allergic forms, several allergens have been identified in the citrus family, the main one being limonene, but no noteworthy cross-reactivity has been identified. However, a case of fruit protein contact dermatitis has been described, showing sensitization to other fruits, such as kiwi, avocado, pineapple and apple. There are several molecules responsible for phototoxic reactions and mainly belonging to the coumarin and furocoumarins families. Reactions related to ingesting the fruit or inhaling pollen from the tree appear to be rare, as there are no known cases reported in the literature. The increasing diffusion of lime in Europe must pay attention to possible adverse reactions due to contact with this fruit, which seem destined to increase in future years. Further importance must be placed on patch tests and on the possibility of using alternative extracts to classic fragrance mixes.Lime ( Citrus aurantiifolia , allergic contact dermatitis (ACD), contact urticaria (CU), protein contact dermatitis (PCD), phototoxic contact dermatitis (PTCD), photoallergic contact dermatitis (PACD), systemic contact dermatitis (SCD) .Citrus aurantiifolia.The etiopathogenesis of these types of dermatitis are different. The aim of this study was to collect and review the published studies and cases of adverse reactions to C. aurantiifolia, below we focus on the analysis of the potentially toxic components responsible for the clinical manifestations to lime.In order to understand the pathogenesis of adverse reactions to Citrus aurantiifolia contains high levels of cumarins and furocoumarins [Citrus aurantiifolia oil contains a high number of monoterpenes (83.93%), in particular d-limonene (40.92%) and Citral (27.46%) are the most relevant [oumarins . A gas ctral 27.4% are theThe main cumarins and furocoumarins contained in the peel and flash of Citrus auratiifolia are 5,7-dimethoxycumarin (Limettin), Psoralen, 5-Methoxypsoralen (Bergapten), 8-Methoxypsoralen (Xanthotoxin) and Isopimpinellin. In addition to Rutaceae, they are common to other plant families such as Umbelliferae, Moraceae, Cruciferae and Ranunculaceae.Coumarins represent a heterogeneous group of natural heterocycles characterized by high chemical diversity Fig. . All nat.Furocoumarins are natural compounds composed by a basic coumarin structure and a furan ring Fig. ; they arThe levels of furocoumarins are higher into peel than into flesh of citrus fruit .The main manifestation due to coumarins and furocoumarins is phytophotodermatitis, a phototoxic reaction.On the skin surface these compounds are excited into a reactive state after exposure to UV radiation and causes a direct toxic effect . These pAt the same time there is an abnormal melanocytes response to UV rays, with an increase of melanosome production of pigment in the oxygen-independent reaction and a melanocytes injury, with pigments’ release in the oxygen-dependent reaction . These rNot all cumarins and furocoumarins have the same phototoxic action..Limettin is not phototoxic at 1% on human skin ..Psoralens (linear compound) are more phototoxic than angelicin (angular compound) , in partd-Limonene, l-Limonene and the racemic mixture dl-Limonene . In one of the case reports, a clear correlation between lime and phytophotodermatitis cannot be demonstrated, as the patient reported that she ingested the lime and painted her face with pigments derived from an unidentified plant root. Subsequent lesions appeared in the same places of application of the pigment .Regarding the allergic reaction to lime, the  reported affect female sex, with average age of 46.5 years old Table .Table 2CPhytophotodermatitis are cutaneous manifestations, due to phototoxic reactions induced by the exposure to sunlight after contact with some species of plants. The molecular components responsible of this clinical manifestation are coumarins and furocoumarins, contained in the plant families of Rutaceae , Umbelliferae , Moraceae (fig), Cruciferae (mustard) and Ranunculaceae (buttercup).The first study dates back to 1993, when Goskowicz et al. described 5 cases of phytophotodermatitis caused by contact with lime in children. They presented blistering eruptions of the photo-exposed areas with streak or finger-print patterns. All of them ate, squeezed or cut limes and the manifestations had risen from a few hours to 24 h after exposure to the sun. Sometimes the rash was painful, and it later turned into hyperpigmented macules, which resolved within some months . Ganesh In Fig. To make differential diagnosis it would be worth executing a punch biopsy of the lesions, which was performed in only one case .The allergic contact dermatitis (ACD) is an inflammatory reaction mediated by T-cells in patients previously sensitized to an allergen.Allergens are low molecular weight chemicals, also called haptens. These molecules must be bound to a carrier protein to become immunogenic .Clinical manifestation occurs 24 to 96 h after contact, in the site of contact, with the appearance of a lesion with well-defined edges that can spread locally or at a distance .In Fig. Lime and other Citrus fruit can cause ACD.D-limonene is the main constituent of peel oil from citrus fruit and experimental studies proved that the handling of the peel oil forms oxidation products like limonene hydroperoxides, that is an allergenic substance . GeranioSwerdlin et al. described a case of lime ACD in a 54 years-old female. Her work consisted in cutting, squeezing lemons and limes, washing dishes, and mixing drinks. She described having itchy, burning, and dry hands daily. The clinical examination showed xerotic hyperlinear palms with rough erythematous plaques on her palms and volar fingers and in most of her web spaces. Her dorsal hands and wrists were spared. Her right hand was more involved than her left hand and these manifestations improved during weekend and vacations.Her history was positive for allergic rhinitis, not for asthma and eczema. The patch test was positive for lime peel exocarp and endocarp but negative for d-limonene .Thomson et al. reported a case of a 52-years old female, who used sucking lime after drinking her gin tonic. She had eczematous eruption on the lips, left corner of the mouth and left chin in the last 4 months. Her history was positive for mild hand eczema and eyelid dermatitis. The patch test performed was positive for myroxylon pereirae, fragrance mix, santolite resin, geraniol 2%, geranium oil, rose oil Bulgarian and lime peel .The last case has been reported by Cardullo et al. who described a case of a 54 years-old female, bartender, who cut and squeezed citrus fruit . She had severe hand dermatitis, which improved during vacations. They performed 3 sets of patch tests: she was positive to fragrance mix 16% (+++) in set 1, positive for lemon, lime and orange peel (++), but negative for lime, orange and lemon juices in set 2. In the last set of patch tests she was positive for citral 2% and 5%, but negative for d-limonene 5% .Protein contact dermatitis (PCD) is an allergic skin reaction which occurs due to the contact with some proteins . It was described for the first time by Hjorth and Roed-Petersen in 1976 .It’s difficult to distinguish it from allergic contact dermatitis .Typically, it presents itself on clinical observation as a form of chronic dermatitis, mainly localized on the backs of the hands and on the fingers and often associated with paronychia .A clinical feature is the immediate appearance of itching, erythema, or blisters, after contact with the responsible protein.Patch-test is typically negative, conversely, the skin-prick test and the scratch test results are positive. The pathogenetic mechanism is not yet well understood; it could be a combination of a type I and a type IV hypersensitivity reaction Fig. . Alterna. The main diagnostic suspicion is protein contact dermatitis but were made other differential diagnoses like contact urticaria caused by latex and fruit or by corn, atopic eczema, and Immediate hypersensitivity to wheat.A 28 years-old pastry chef had burning sensation and itch of the hands after contact with lime, pineapple, avocado, drupe, flour and cucumber. Her dermatitis got worse in the last 3 years and spread to face, foreharm, extremities and chest. She referred daily contact with lime zest. Furthermore, she had allergic rhinitis and asthma. Both cutaneous and respiratory symptoms improved during weekends and vacations. She had a positive history of atopic dermatitis, confined to her flexures and well controlled. Prick test was positive for dust mites, cat and horse epithelium, lime, apple, avocado, pineapple, and kiwi. All Patch tests performed gave negative results. RAST  was positive for latex, hazelnut, almond, lemon, peach, wheat, corn, kiwi fruit, avocado and macadamia nuts . The maiCitrus aurantiifolia fruit. There was no evidence of adverse reactions after both ingestion of the fruit or inhalation of tree pollen.Literature analysis revealed only contact reactions to All clinical manifestations were cutaneous with no involvement of other organs.There are different ways to encounter this fruit, like squeezing, cutting, or picking. In addition to the common use of lime, like in alcoholic and non-alcoholic beverages, cosmetics, and food preparations, it is important to focus on other ways to use this fruit, for example as a natural remedy , 31.Citrus aurantiifolia for occupational reasons, such as bartenders, gardeners, or pastry chefs.Many of the cases described concern patients exposed to C. aurantiifolia contain coumarins and furocoumarins, the main responsible of phytophotodermatitis. The features of the lesions make this type of dermatitis easily misdiagnosed. Other differential diagnoses must be considered, such as physical violences or burns. Most reported cases have happened in tropical areas, so the duration of the exposure to UVA and the latitude at which it occurs must be considered as significant environmental factors. Ji Young Choi et al. suggest the importance of the energy level of the UVA ray: they have made up a skin provocation test applying Finn chambers full of lime extract on the volunteers’ back and after two hours they have irradiated with various doses of UVA; after 3 days this test has showed the erythema appeared only on skin exposed to an energy level as low as 10 J/cm2 [Adverse reactions to lime are mainly phytophotodermatitis, few cases are allergic contact dermatitis (ACD) and only one case is described as protein contact dermatitis (PCD). The etiopathogenesis of phytophotodermatitis is well known. Both flesh and peel of 10 J/cm2 .Therefore, to make a correct diagnosis, a careful medical history cannot be ignored: employment status, history of recent trips to tropical areas and consumption of drinks containing lime are fundamental to collect. Age and sex factors should be taken into consideration, as phytophotodermatitis appear to be more common in young women.In men it seems to be less frequent and occurs mainly in the pediatric age. However, data related to the cases of phytophotodermatitis in males are limited Fig. .Regarding allergic reactions to lime, type IV hypersensitivity is the main pathogenetic mechanism (ACD). However, lime could be responsible for protein contact dermatitis (PCD), which has a different pathogenetic mechanism than ACD, not yet well known. To discriminate between the two forms is needful to know the timing of onset of the lesions. These usually appear 24–96 h after exposure, in the contact point and with possible remote localization, in ACD. Late onset of lesions and the pathogenetic mechanisms justifies patch test as the main diagnostic test in ACD, that is typically positive. In PCD there is an acute phase with erythema, itching and blisters appearing immediately in the contact point; secondly, we can find signs of eczematous chronic dermatitis. The negativity to the patch test with a positivity to the skin prick test is a feature of PCD, which suggests that the pathogenetic mechanism seems to be a combination of immediate type I and delayed type IV hypersensitivity.D-limonene is recognized as the major allergen of the genus Citrus but in the 3 cases reported in our analysis patch tests were negative to D-limonene and positive to Geraniol and Citral, that are minor allergens .According to literature there is evidence regarding the greater sensitivity of the patch test performed with Citral or its singular components  and the oxidized forms of Limonene and Geraniol compared to baseline fragrance materials currently used , 15.Regarding PCD, the only case reported is controversial. The onset of clinical manifestation and the diagnostic tests argue in favor of this diagnosis, but the patient had multiple sensitizations and she was exposed to different allergens at the same time; so it’s difficult to identify the real culprit of the allergic reaction. In the end, chronic exposure to multiple allergens in an occupational environment confuses the clinical scene.Increasingly widespread use of lime in the world , attention must be paid to the potential increase in adverse reactions to lime in the coming years.In some people and in specific environmental conditions lime can cause skin reactions such as phytophotodermatitis, allergic contact dermatitis (ACD) and protein contact dermatitis (PCD).A thorough history and examination can help identify lime as a cause of these clinical skin manifestations enabling implementation of successful avoidance strategies. Anamnestic collection must include questions about occupational exposure, contact with lime during recent trips in tropical areas, timelapse between contact with lime and onset of clinical manifestations, different and unusual ways to encounter this fruit.After all, it could be useful to evaluate the use of alternative preparations to those commonly used in patch tests. To increase the sensitivity of this diagnostic test, it would be more appropriate to use individually the molecular components .


Over the years, questions have been raised over R&D costs of new medicines. The aim of this study is to conduct a landscape review of the (drivers of) R&D costs of a new medicine derived from the peer-reviewed and grey literature. Included studies have drawn data either from confidential company surveys or from publicly available company financial statements, in addition to accessing the literature and medicine information databases. Although there were differences in methodology, parameter values, samples and time periods between studies, estimates of R&D costs per new medicine (accounting for the cost of failures) ranged from US$944m to US$2,826m (adjusted to 2019 prices). The evidence also suggested that R&D costs per new medicine have increased over time. A few studies have broken down total costs and showed that clinical development accounts for 50-58% of R&D costs per new medicine. R&D costs were influenced by costs of discovery and pre-clinical development, costs of clinical development, cost of capital, company and product profile. Finally, cost estimates are likely to be dynamic as the biopharmaceutical industry and the broader environment continue to evolve. There has been a long-standing interest in and discussion about the costs to research and develop a new medicine. It is useful to gain insight into those costs as such information may serve to get an idea of how much investment is required to research and develop a new medicine, to use in the assessment of R&D productivity of the biopharmaceutical industry, to inform the debate about medicine prices, or to explain trends in R&D approaches and in the industrial landscape , 2.In the literature, two reviews were published around a decade ago, but these reviews did not include more up-to-date empirical studies estimating R&D costs of a new medicine , 3. ReceA search was carried out in PubMed to identify studies which estimated R&D costs (and their components) of new medicines based on empirical data. Articles that commented on R&D costs, but did not provide new estimates were excluded. Studies were considered if they had been published between 2000 and 2020, as earlier cost estimates are unlikely to reflect current R&D processes. The search was restricted to English-language studies. Also, the grey literature was searched in Google (Scholar), including empirical studies conducted by consultancy agencies . Finally, the snowballing method was applied, identifying additional studies from the bibliography of included studies.A data extraction sheet compiling information about the objectives, design, results, conclusions and limitations was completed for each included study. For the purpose of comparing results between studies, all cost estimates were adjusted to 2019 prices using the World Bank GDP deflator .Our search of the peer-reviewed literature identified three literature reviews (cfr. supra), eight empirical studies on R&D costs of a new medicine which derived their data from confidential company surveys in addition to the literature and medicine information databases, and two empirical studies which derived their data from publicly available company financial statements. The grey literature generated a series of Deloitte reports on R&D costs based on company financial statements. Another consultancy report by Bain and Company provided cost estimates, but was excluded because no details of the underlying methodology were reported . Data exIn 2003, a study calculated the pre-tax R&D costs per new medicine (accounting for the cost of medicine failures) based on a random sample of 68 medicines  developed in-house by 10 multinational companies  and geneAlthough there are differences in methodology, parameter values, samples and time periods between studies, A few studies have also broken down mean capitalised R&D costs per new medicine into costs for discovery and pre-clinical development and into costs for clinical development see . These sTwo more recent studies have calculated the pre-tax R&D costs per new medicine (accounting for the cost of medicine failures) based on publicly available company financial statements, thus allowing to validate and replicate their cost estimates. Both studies included in-house developed medicines as well as acquired medicines. One study focused specifically on ten oncology medicines (nine of which had orphan medicine designation) from small companies . The othThe study focusing on oncology medicines found that mean capitalised R&D costs per new medicine amounted to US$944m (adjusted to 2019 prices) . Also, cBased on the financial statements of the 12 largest biopharmaceutical companies (in terms of R&D investment in 2009), public sources and medicine information databases, Deloitte has calculated R&D costs per new medicine (accounting for the cost of medicine failures) since 2010 . AdditioAs visualised in In addition, R&D costs need to be “capitalised”. This implies that the estimate is adjusted using a cost of capital, which reflects the return required for an investment today that generates revenues in the future. Capitalisation has a considerable impact: the cost of capital made up 33-51% of total costs per new medicine in four studies , 11, 13.Furthermore, the literature indicates that R&D costs per new medicine depend on the product class, whether the medicine is developed in-house or is acquired, and company characteristics .Finally, the broader industrial, regulatory and policy environment influences R&D costs of new medicines. For instance, the literature generates pre-tax estimates and, hence, does not consider deductibility of R&D expenditure for taxation purposes . Cost esAlthough a few studies break down R&D costs of new medicines into costs associated with different drivers , 11, theEstimates of R&D costs per new medicine derived from the peer-reviewed and grey literature vary between US$944m and US$2,826m (adjusted to 2019 prices). The evidence also points toward an increase in these costs over time. Any cost estimate needs to be interpreted in the context of and is specific to the underlying methodology, sample and time period of the study. Estimates are also dynamic: given that the biopharmaceutical industry and the broader environment continuously evolve, the costs to research and develop a new medicine change. Future research would benefit from greater accessibility and transparency of R&D cost data and from improved methodologies to attribute costs of discovery and pre-clinical development to individual medicines.SS developed the idea and design of this study, carried out the literature review, and wrote the manuscript. IH critically reviewed the manuscript. Both authors contributed to the article and approved the submitted version.This research was funded by the European Federation of Pharmaceutical Industries and Associations (EFPIA). The sponsor had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the paper; and in the decision to submit the paper for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To analyze the real-time infection dynamics, we described the generation of a mouse-lethal recombinant H9N2 AIV, an influenza reporter virus (VK627-NanoLuc virus) carrying a NanoLuc gene in the non-structural (NS) segment, which was available for in vivo imaging. Although attenuated for replication in MDCK cells, VK627-NanoLuc virus showed similar pathogenicity and replicative capacity in mice to its parental virus. Bioluminescent imaging of the VK627-NanoLuc virus permitted successive observations of viral infection and replication in infected mice, even following the viral clearance of a sublethal infection. Moreover, VK627-NanoLuc virus was severely restricted by the K627E mutation in PB2, as infected mice showed little weight loss and a low level of bioluminescence. In summary, we have preliminarily established a visualized tool that enables real-time observation of the infection and replication dynamics of H9N2 AIV in mice, which contributes to further understanding the mechanisms underlying the pathogenic enhancement of H9N2 AIV to mice.H9N2 avian influenza viruses (AIVs) continuously cross the species barrier to infect mammalians and are repeatedly transmitted to humans, posing a significant threat to public health. Importantly, some H9N2 AIVs were found to cause lethal infection in mice, but little is known about the viral infection dynamics  Sincein vitro or in vivo studies, such as the screening of antiviral compounds or neutralizing antibodies, the development of vaccines, or the identification of host factors  or Renilla luciferases  by reverse genetics. VK627-NanoLuc virus possessed similar virulence and replicative capacity in mice to its parental virus. Bioluminescent imaging of the VK627-NanoLuc virus allowed continuous observations of viral dynamics in infected mice. Overall, these results provide an effective tool to study the real-time infection dynamics of H9N2 AIV in mice, facilitating the exploration of the mechanisms of enhanced virulence of H9N2 AIV to mice.Based on the strategy of the NS segment generating a reporter virus, we produced a recombinant H9N2 AIV expressing the NanoLuc luciferase (VK627) was previously described (BsmBI restriction sites. Madin-Darby canine kidney (MDCK) and Human embryonic kidney cells (HEK293T) were maintained in Dulbecco's modified essential medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C in 5% CO2.The eight-plasmid reverse genetics system for H9N2 avian influenza virus A/Chicken/Guangdong/V/2008 (Vescribed . The nonescribed . The NS-K627-NanoLuc virus was rescued by reverse genetics techniques (K627 and the NS-NanoLuc-NEP segment were transfected into HEK293T cells in six-well plates by using Lipofectamine 2000 (Invitrogen). After 6 hours, the medium was replaced with Opti-MEM (Gibco) including 1 μg/ml L-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK) treated trypsin (Sigma). 48 h later, the HEK293T cells were resuspended and harvested in the medium, and the mix was injected into specific pathogen-free (SPF) chicken embryonated eggs. Then VK627-NanoLuc virus was confirmed by performing a hemagglutination assay. The VK627E-NanoLuc virus was rescued by a single K627E mutation in the PB2 protein of VK627-NanoLuc virus with the same protocol. VK627 parental virus was as a control, which contained PB2 residue K627 and was lethal to mice in our previous study (Vchniques . Brieflyus study .K627-NanoLuc virus) was labeled as P0 and then passaged in SPF embryonated eggs for three generations . RNA obtained from viral stocks (P0-P3) was extracted with the RNeasy minikit (Qiagen) as directed by the manufacturer. The sequences of the NanoLuc gene in the viral stocks were confirmed by reverse transcription-PCR (RT-PCR) reported by Hoffmann et al.  at 24 hours post-infection (hpi). The protein samples were followed to SDS/PAGE and transferred to a nitrocellulose membrane. NS1 protein and NS1-NanoLuc fusion protein were detected by immunoblotting with a mouse polyclonal anti-NS1 antibody  and the viral nucleoprotein (NP) was detected by immunoblotting with anti-NP antibody , and followed by IRDye 800CW, goat anti-mouse IgG . The membrane was imaged using an Odyssey infrared imaging system .MDCK cells grown in six-well plates were infected with V2. Culture supernatants were collected at 12, 24, 36, and 48 hpi. Titers were determined by performing 50% tissue culture infective dose (TCID50) assays on MDCK cells.MDCK cells grown in six-well plates were infected at a multiplicity of infection (MOI) of 0.001 at 37°C, as described previously . One houn = 5/group)  were anesthetized with isoflurane and inoculated intranasally with VK627E-NanoLuc and VK627-NanoLuc virus at a dose of 106 EID50/50 μl. The body weight and survival of mice were monitored daily for 14 days. Mice were humanely euthanized when they lost more than 25% of their body weight.Pathogenicity of Viruses: 5-week-old female BALB/c mice  were anesthetized with isoflurane and inoculated intranasally with VK627 virus and VK627-NanoLuc virus at doses of 103, 104, 105, or 106 EID50/50 μl. The body weight and survival of mice were monitored daily over a period of 14 days. Mice were humanely euthanized when they lost more than 25% of their body weight. LD50 values were calculated by the method of Reed and Muench. To detect the viral replication in the lungs of mice, another three mice from the group of 106 EID50/50 μl were euthanized at 5 days post-infection (dpi) and viral titers in the lungs were determined by an EID50 assay.Determination of LDThe same mouse from each group was imagined at the appointed time by using an IVIS imaging system. Briefly, infected mice were anesthetized with isoflurane and 100 μl Nano-Glo reagent  was injected into retro-orbitally of mice. The Living Image software was used for image acquisition and analysis. Flux measurements were automatically calculated from the signal of mice. All data of composite images used the same scale.n ≥ 3) unless otherwise noted. Multiple comparisons were performed by using an unpaired t-test in the GraphPad Prism software (GraphPad Software Inc.). Significance is defined as P < 0.05 and is indicated with an asterisk (*).Data represent means ± standard deviations (SD)  was rescued (marked as P0). The amino acid at position 627 of the PB2 protein on both VK627-NanoLuc virus and parental virus VK627 is lysine (K).The NS segment of the influenza virus encodes NS1 protein and NEP protein produced from unspliced mRNA and spliced mRNA, respectively . It has nslation . By usinK627-NanoLuc virus (P0) was propagated in SPF embryonated eggs for three generations . RT-PCR and Sanger sequencing were performed on RNA obtained from viral stocks. As shown in K627-NanoLuc viruses. To detect the expression of NS1-NanoLuc fusion protein, Western blot analysis was performed in MDCK cells  was detected. In VK627-NanoLuc virus-infected cells, the band corresponding to NS1-NanoLuc fusion protein (~ 43 kDa) was observed. These results showed that the VK627-NanoLuc virus expressed NS1 protein and NanoLuc luciferase as a fusion protein.The stability of the NanoLuc reporter gene was tested following amplification in SPF embryonated eggs. The rescued VCK cells . In the K627- NanoLuc virus affected the viral replication in culture, we compared the growth kinetics of VK627-NanoLuc virus and parental virus VK627 in MDCK cells .Vur study , 37. To t effect . In the nfection . In the survived . Accordi6 EID50/50 μl at 5 dpi. The result showed that the VK627-NanoLuc virus replicated efficiently in the lungs of mice, with mean titers reaching 4.33 lgEID50/200 μl, slightly lower than VK627 virus (4.83 lgEID50/200 μl)  . Combinein vivo imaging of reporter viruses is useful to study the viral infection dynamics for different viruses, including influenza A virus  to analyze the real-time infection dynamic of H9N2 AIV in mice. Our work has demonstrated that the virulence and replicative capacity of the VK627-NanoLuc virus in mice were very close to its parental virus. Furthermore, in vivo imaging of our reporter virus was able to visualize viral replication in mice models.H9N2 AIVs have been distributed in various avian species worldwide and are repeatedly spread to mammals. In general, H9N2 infections in poultry or human are mild. However, some H9N2 AIVs were reported to cause lethal infection in mice –22. To bin vivo, so large insertions or a simple replacement of reporter genes may severely attenuate viral replication. Most importantly, the inappropriate location of the reporter gene insertion can interfere with viral packaging signals and subsequently affect virus assembly. In our reporter virus, a small NanoLuc reporter gene was inserted into the middle of the NS segment to circumvents the repetition of packing signals. In the same strategy, various recombinant influenza viruses carrying foreign genes in their NS segments have been successfully used in vitro and in vivo , China Postdoctoral Science Foundation Funded Project (2021M691082), and Young Scholars of Yangtze River Scholar Professor Program .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In spite of a current increasing trend in the development of miniaturized, standalone point-of-care (PoC) biosensing platforms in the literature, the actual implementation of such systems in the field is far from being a reality although deeply needed. In the particular case of the population screenings for local or regional diseases related to specific pathogens, the diagnosis of the presence of specific antibodies could drastically modify therapies and even the organization of public policies. The aim of this work was to develop a fast, cost-effective detection method based on the manipulation of functionalized magnetic beads for an efficient diagnosis of hypersensitivity pneumonitis (HP), looking for the presence of anti-pigeon antigen antibodies (APAA) in a patient’s serum. We presented a Diagnostic Biosensor Method (DBM) in detail, with validation by comparison with a traditional high-throughput platform (ELISA assay). We also demonstrated that it was compatible with a microfluidic chip that could be eventually incorporated into a PoC for easy and broad deployment using portable optical detectors. After standardization of the different reaction steps, we constructed and validated a plastic chip that could easily be scaled to high-volume manufacturing in the future. The solution proved comparable to conventional ELISA assays traditionally performed by the clinicians in their laboratory and should be compatible with other antibody detection directly from patient samples. Hypersensitivity pneumonitis (HP) is a complex syndrome caused by exposure to a large variety of organic particles, small enough ~5 µm) to reach pulmonary alveoli ,2. HP is µm to reThe diagnosis of HP requires the integration of multiple domains, including radiological, functional, and bronchoscopic/histopathological findings, but is particularly challenging in the absence of an identifiable exposure. In this context, antigen identification is critical to tackling HP. Measurement of specific immunoglobulin G antibodies (IgG) in a patient’s blood serum is an important method used to identify an inciting antigen and is proposed as a diagnostic test for HP ,8,9. TheRecent combined progress in microtechnology and precise protein immobilization on a variety of materials allowed for the development of microfluidic platforms designed as low cost easily operated point-of-care (PoC) biosensing systems offering a broader availability in locations where the use of costly and complex instruments is not an option ,12,13. Compared with more conventional existing techniques, these PoC systems can integrate high-sensitivity detection methods with simple readouts and data logging platforms to provide rapid and cost-effective solutions for long-term monitoring and diagnostics ,15. LargOne of the possible simple solutions to integrate ELISA platforms in microfluidics chips that may help to overcome these restrictions consists in the use of magnetic beads . Their sThe aim of this work was to design a biosensing technique compatible with a simple, low-cost PoC platform to detect the presence of human IgG antibodies in patient’s serum samples of avian-antigen-induced HP, using pigeon antigen sera coupled to commercial magnetic beads. After validation of all the different reactions of the method in microwells at the lab bench level, a final microfluidic chip was designed and tested for future use in PoC. For this purpose, a motorized actuated platform was also fabricated to precisely manipulate functionalized magnetic beads throughout biological samples and to bind specifically the desired antibodies inside a microfluidic chip that allowed a simple optical readout in an ELISA-like fashion. The method, called Diagnostic Biosensor Method (DBM) in all the following, was successfully validated using patient’s samples, and preliminary results compared well with a traditional ELISA performed in a hospital clinical laboratory, which is currently used to diagnose HP.We used samples from 30 HP patients of the National Institute of Respiratory Diseases (INER). HP was diagnosed as previously described, using clinical symptoms, history of antigen exposure, and specific antibodies against avian proteins measured by conventional ELISA, pulmonary function tests, high-resolution computed tomography, an increase in lymphocytes in bronchoalveolar lavage, and/or surgical biopsy ,2. The sThe method used in this work followed a published procedure  in which2SO4 1 mol/L. The absorbance was read in a microplate spectrophotometer at 492 nm with a correction factor at 595 nm.The conventional ELISA was performed with a microtiter plate coated with the pigeon sera diluted in a carbonate buffer, incubated overnight at 4 °C, and washed 3 times with PBS-Tween 20. Plates were then blocked with PBS-Tween containing 0.5% bovine serum albumin (BSA) for 1 h at 37 °C on a horizontal orbital microplate shaker and washed 3 times with PBS-Tween 20. Patient sera were diluted 1:6250 for the IgG assay in PBS-Tween containing 0.5% BSA. Under these conditions, each sample was studied in triplicate, and plates were incubated for 1 h at 37 °C with shaking. Purified goat anti-human IgG (Fc specific) conjugated with horseradish peroxidase  was added and incubated for 1 h at 37 °C with shaking. Finally, orthophenylenediamine  was added and incubated at 37 °C with shaking for 10 min, and the reaction was stopped with HSerum samples were measured simultaneously using conventional ELISA assay versus the detection with the developed method of magnetic beads. 2) reaction was employed for optical detection by fluorescence microscopy and quantification of the signal emitted by the Ab2-marked beads.The Diagnostic Biosensor Method presented here was based on the use of functionalized magnetic beads as elements of biological recognition to detect biomolecules of interest contained in a sample using an immunodetection method. The detection procedure consisted of three steps, depicted in This DBM was versatile and could be used in either reaction microtubes, 96 well plates, as well as a transparent microfluidic chip. All the reactions were first standardized and optimized in 1.5 mL microtubes and non-treated polystyrene flat-96-well assay plates prior to the final tests inside the system of the microfluidic chip. This diagnostics validation with patient samples was performed inside ad hoc microfluidic chips see . Although different concentrations of magnetics beads and volumes of reaction solutions were used, the same ratio of proteins , with respect to the number of beads, was maintained in each test platform. We herein related all the protocol steps to 80 K magnetic beads, number of beads used in the microfluidic chip .® Tosylactivated M-450 Invitrogen, Carlsbad, CA, USA), basing the protocol on the manufacturer’s recommendations. Beads were resuspended by vortexing ; the desired amount of beads was transferred to a 1.5 mL low binding microtube  and washed with 500 μL of Buffer 1 . Then, beads were resuspended in 500 µL of a PS solution in Buffer 1  and it was incubated for 24 h (16 h and 24 h were tested) at room temperature while shaking in a HulaMixer  setting orbital 15 rpm/10 s; reciprocal 20°/15 s; vibro 1°/1 s, horizontal, allowing tilt and rotation of microtubes. Afterward, two washes of 5 min each were performed with 500 µL of Buffer 2 at 2–8 °C  with a HulaMixer agitation. In order to block the remnant tosyl groups, beads were then resuspended with 500 μL of Buffer 3  and incubated for 4 h in Hulamixer agitation at 37 °C . After blocking the incubation, a final wash with Buffer 2 was performed, as described above. The beads were resuspended in a vortex (30 s) at the desired volume (10 µL/80 K beads) in PBS supplemented with 0.01% Tween at pH = 7.4  and immediately transferred (10 μL) to the corresponding reaction platform. As described in The beads functionalization consisted of a covalent coupling reaction between the amino and sulfhydryl functional groups of the pigeon antigen and the tosyl groups of magnetic beads  sample with different APAA concentrations . For all the samples with unknown concentrations, 0.08 μL patient serum/80 K beads were used for diagnostic probes. The reaction was carried out on the different platforms as follows. Inside the chips, the beads were displaced using a motorized actuator platform  were eliminated on the chip: instead, to minimize the hauling of undesired free antibodies caused by the dragging force of the beads during their movement from one well to another, the chip design included intermediate wells with PBS-Tween 0.01% .® 488-conjugated AffiniPure Alpaca Anti-Human IgG (H + L) secondary antibody  was performed, and different concentrations were tested to determine the optimal one . The procedure ran as follows: in a chip, the beads were mobilized to the αHIgG-488 well ; in microtubes and plate αHIgG-488 solution was added  and resuspended by the vortex. Incubated  at room temperature while shaking . Inside the chip, the beads were just mobilized to the optical reading well , passing through the intermediate washing well. In microtubes and plates, PBS washes were performed as described in Step 2, and the beads were resuspended in PBS  for the final optical readout. Finally, to detect and quantify the bounded IgG APAA, a reaction with Alexa FluorThe magnetic beads processed in microtubes and plates were observed with the epifluorescence microscope TCS-SP8 . After mixing the sample, a 10 μL drop was placed on a slide, and a magnet was used to spread the beads on a single focal plane. The beads were excited at 488 nm, with the GFP filter . To visualize the entire sample, a 10× magnification was used with an exposure time of 4 s and a gain of 10. The final xy resolution was 1.118 µm. Several images per sample were taken and exported in tiff format for analysis.Fluorescence intensity was calculated per sample through the computing of the fluorescence intensity at three different levels: bead level, image level, and sample level. At the bead level, a Laplacian of Gaussian filter (LoG filter) was used for the beads segmentation and detection in the images. After precise spatial location, the fluorescence intensity of each bead was calculated by averaging the values of the set of pixels constituting it. The minimum and maximum intensity levels, the number of identified beads, and the average fluorescence intensity per image was obtained. Besides the average, two additional values were computed: the 75th percentile and the sum of all N values above the 25th percentile divided by N (the weighted sum). Finally, the fluorescence intensity values of all the images related to a single sample were used to compute the same values . In all averaging cases, an associated standard deviation was computed, including data used for the weighted sum. A detailed description of the method used to detect the beads in the images was discussed in the An ad hoc microfluidic chip with a motorized actuated system was conceived to precisely manipulate magnetic beads with magnets inside microwells. It could be regarded as the first functional prototype, a promising progress towards a standalone reader and actuator for PoC implementation. The motorized system was constructed with aluminum extrusion, stepper motors, microcontrollers (Arduino and Teensy), laser cut, and 3D printed custom parts; all of these materials were readily available and relatively inexpensive. The final design and fabrication procedure were set after a careful standardization of bead displacement and washing steps that was described in the 2 for five minutes. This step activated the surfaces, increasing their wettability for optimal adhesion and sealing [In order to obtain a hermetically sealed microfluidic chip from the perforated PMMA layer, two lids of the same dimensions were necessary. For this laboratory prototype version, a glass slide  was used for the top lid, and a 380 µm thick slab of flat, pristine PMMA was cut with the same dimensions to serve as chip bottom . Note that a thinner piece was selected for this bottom lid to guarantee a better interaction between the magnets and the beads. The two PMMA pieces were then carefully assembled: the two sheets were exposed to UV inside a longwave UV crosslinker with a 254 nm lamp  with an energy of 999,900 microjoules per cm sealing . After t sealing  was usedTo avoid undesired internal mixing between wells, the connecting microchannels were filled with 1.6 µL of mineral oil  through low retention micropipette tips using the small filling ports located at the center of each channel . The filA custom-made automated platform was also constructed for a robust and more reproducible manipulation of the magnetic beads inside the microfluidic chip. The motorized setup shown in  allowed To increase reproducibility and favor the interactions of beads with the complete volume of the sample (well 2) and the anti-human IgG APAA-αHIgG-488 (well 4), a vertical stirring routine was programmed during the whole reaction time . For thaFinally, at the location of the detection well (well 6), a simple combination of vertical and lateral movement was used to impose a flat spreading of the beads across the bottom surface of the well . This coAfter the beads were spread in a controlled way at the bottom of the detection well, the chip was carefully removed from the automated platform and placed on an ad hoc holder for posterior image acquisition on an inverted fluorescence microscope , focusing the beads inside the detection well using bright field microscopy. Once focused, three different regions of interest with low bead agglomeration were photographed using filters: GFP  and CY5  with a 20X magnification, an exposure time of 164 milliseconds, and a gain of 15. The images acquired were analyzed using the procedure described in t-test was used to calculate statistical significance for p-value < 0.05 was considered to be significant. A paired t-test was considered for t-test was used for A student’s To optimize the DBM before applying it directly on the microfluidic chip, different parameters of the three reaction phases described before were optimized. First, we determined the concentration of PS for functionalization and the incubation time that allowed the capture of the greatest amount of IgG APAA in the samples (method step 1). The fluorescence intensity obtained with functionalized beads with PS solutions at different concentrations  and incubation times (16 h vs. 24 h) were compared. In the DBM steps 2 and 3, serum antibodies from an HP-positive patient (18.3 μg/80 K beads) and secondary antibody αHIgG-488 (1.7 μg/80 K beads) were used; both reactions had incubation times of 2 h and with 1.5 mL low-binding microtubes as the reaction platform. The results depicted in Once the optimal functionalization conditions were defined , the optimal αHgG-488 concentration required to obtain an acceptable level of detection was determined, referred to as saturation in fluorescence intensity, allowing to discern the range of IgG APAA in the patient serum. For this purpose, tests in microtubes were carried out in which the concentration of the HP-positive patient serum  was maintained, but different concentrations of αHgG-488  were evaluated. The results showed that the saturation in fluorescence intensity was reached at concentrations of 0.6 and 1.3 μg/80 K beads B.p = 0.004 for DBM), and saturation of the signal was observed at around 73.3 ng/μL . Representative microscopy images of the magnetic beads functionalization and reaction are also illustrated in Once DBM step 1 and 2 were settled, in order to determine the sensitivity, a dose-response curve using different concentrations of HP-positive patient serum  was performed using microtubes. To compare with the ELISA, the following equivalent concentrations were used, respectively: 5.5, 9.2, 12.2, 17.6, 36.7, 73.3, 220 ng/μL, in 500uL. Furthermore, the comparison was made with the usual employed ELISA using the same patient serum C. The seTo verify whether there was a non-specific binding of patient serum or αHIgG-488 to the non-functionalized and functionalized magnetic beads, controls with different conditions on the three steps of the DBM were performed in microtubes and compared to a full reaction that was PS-functionalized beads, patient sample and secondary antibody αHIgG-488 . It coulAccording to the literature, the detection limit signal of an analytical method should be greater than three times the standard deviation of the blank measurement : to takeTo validate the DBM for HP, by detecting the presence of human IgG anti-pigeon antigen antibodies in sera, we tested 30 serum samples of HP and controls (positive and negative). The reactions of this test were carried out in microtubes: beads were functionalized with PS —1.3 μg/80 K beads αHgG-488, and 0.08 μL patient serum/80 K beads were used—both reactions with 2 h of incubation time. As a negative control, the functionalized beads reacted with PBS instead of the patient’s serum and later with αHgG-488. The fluorescent intensity and the diagnostic obtained were compared against a conventional ELISA. The results shown in p < 0.0001; A Receiver Operating Curve (ROC) was executed to assess the area under the curve (AUC) for the DBM as a diagnostic tool for HP. The AUC was very high (0.9667) and the cutoff point related to the highest specificity (96.67%), and sensitivity (100%) for the DBM variable was 55.80. The crosstabulation showed 96.67% sensitivity and 100% specificity, a positive predictive value of 96.67%, a negative predictive value of 100%. In order to determine the level of agreement between the ELISA and the DBM, we performed a Cohen’s kappa analysis. The level of agreement between the two diagnostic methods had a kappa value of 0.966  in order to compare between reaction platforms. The conditions of the reactions, for both well plate and chip, were 3 μg/80 K beads PS for 24 h for beads functionalization, 0.08 μL patient serum/80 K beads, and 0.63 μg/80 K beads αHgG-488. Incubation times for the patient sample and secondary antibody were changed from 2 h to 45 min . As seenThe lower band of The aim of this work was to develop a fast, cost-effective detection method for efficient diagnostics of HP looking for APAA in serum that allowed the future development of both a PoC and a high-throughput platform. In this context, we presented the DBM that, after standardization of the reaction steps, was validated in a plastic microfluidic chip, which can easily be scaled to high-volume manufacture in the future. The use of magnetic beads with an ad hoc motorized platform, made from readily available materials, allowed for a precise reaction and manipulation of the samples up to the optical detection stage; since an integrated standalone detector will be developed in the future, a commercial system was used.® xMap®, LEGENDplexTM, and FIREplexTM; while these methods represented important advances in the detection of multiple analytes for a single sample, they were not readily available. One of the main issues was that they required sophisticated and expensive equipment to obtain the fluorescence readings. The use of this equipment was not easy, and only trained personnel could do so; thus, they were made for large-scale use, limiting their potential use for a PoC device. Other initiatives of the use of magnetic beads on 96 well plates have been published [Commercial magnetic beads ELISA was developed as Luminexublished . In thesThe use of microfluidic devices without the use of magnetic beads has recently been published . These mTherefore, the work carried out here represented the conjunction of the two advantages published so far: the use of magnetic beads as a reaction and transport surface using the base of a competition ELISA and its adaptation to a microfluidic device with a motorized platform that automated the reaction process. Sensitivity and efficiency. Although the positive to negative ratio of the DBM was lower than that of the traditional ELISA, indicating lower sensitivity, the results obtained with the DBM, as demonstrated by ROC and kappa analysis, were in accordance with the measurements made with the ELISA employed on HP diagnosis. As shown in the results, the background noise ratio in this ELISA test was lower than that obtained for DBM. However, it was worth saying that the horseradish peroxidase (HRP) produced a colored, fluorimetric derivative when incubated with a suitable substrate. As its name indicated, it is an enzyme that performs two redox reactions, meaning that the background noise depended on time, temperature, and the handling of the inhibition reaction, so it represented a variable value. The DBM method of detection was fluorescent and, therefore, the noise value did not depend on the handling and was very stable from one test to another.Patients with different anti-pigeon antigen antibody concentrations could be successfully detected and diagnosed. As demonstrated, the DBM could also be adapted for its use in microtubes, well plates, as well as microfluidic chips. One of the main problems of human IgG measurement using ELISA was that whole blood was not employed, and the serum had to be separated and diluted in order to obtain a correct signal. This complicated the diagnostic process and made it useless for the development of a PoC. Our results showed that in the microfluidic chip, the detection reaction was more efficient than the same reaction in plate and microtube, and the results from tested sera with varied antibody concentrations indicated a greater sensitivity. Moreover, fingerstick whole blood samples did not generate any noise signal ; it was Fast and automated detection method, the detection procedure of DBM lasted 1h 30min in a 96 well plate and 1h 10min in the microfluidic device in comparison with the minimum 3 h required for ELISA. The development of the custom-made automated platform for the controlled manipulation of the magnetic beads inside the microfluidic chip allowed the automation of the diagnostics. Therefore, the user would only have to put the sample in the chip well, and the platform would perform the displacement of the magnetic beads until the detection device might be employed, either individually or on a large scale with many samples at the same time, as in clinics and hospitals. In this case, it would limit the constant intervention of staff required in other methods. Remarks on the cost. The costs of performing DBM in American dollars were, for one sample in microtube 4USD, 96 wells plate 2USD, microfluidic chip 3USD, compared to the ELISA´s 6USD cost. In addition, as mentioned above, one of the great advantages of using the DBM on the microfluidic chip was that the user only had to put the sample on the chip well, and the platform automated the reaction process until the detection device might be employed. This represented a reduction in the costs associated with human error due to manipulation, as well as freeing up the user’s time for other matters.Another big difference that influenced the cost was the use of laboratory equipment and material: in this case, the DBM was compatible with different reaction devices; therefore, its results could be read in multiple devices, from an ELISA plate reader to a microscope or a cytometer, among others. Point-of-Care and high-throughput diagnostic, the use of fluorescence as a marking signal was a wide field for which DBM could be used in multiple detections with the same sample, minor changes to the microfluidic chip could be carried out in order to achieve this goal. The chip design might be used for further reading using a fluorescence sensor for on-chip quantification and rapid diagnostics on a PoC implementation. This then needs to be implemented in the future and was outside the scope of the present phase of the project. However, an imaging system may already be employed for diagnostics; an automated program was also developed to be used from micrographs.The DBM designed to detect the presence of human IgG antibodies in patient’s serum samples of avian-antigen-induced HP  proved to be comparable for diagnostic purposes to conventional ELISA assays. The DBM principle was not limited to be used in an exclusive device of reaction. In conjunction with the microfluidic chip and a custom motorized actuated platform, it led to a number of advantages in the performance of the tests such as automation, reduced cost and testing time, the possibility of performing isolated or multiple samples, and no noise signal on a fingerstick whole blood sample. All these advantages, in addition to the use of fluorescent markers, led us to believe in a wide range of applications in biomolecules and equipment detection. We showed the signs of the development of a PoC, whose implementation could be possible in the near future, for biological and clinical applications other than avian-related hypersensitivity pneumonitis. This made it possible to offer a greater availability of analytic tests in places where the use of expensive and complex instruments is not an option.


First,when the cellulose concentration is lower than 0.5 wt %, the dryingprocess comprises three steps and, regardless of the water/ethanolmixture composition, a sharp structural transition corresponding tothe formation of a cellulose II crystalline structure is observed.Second, when the cellulose concentration is higher than 5.0 wt %,a two-step drying process is observed and no structural transitionoccurs for any of the beads studied. Third, when the cellulose concentrationis between 0.5 and 5.0 wt %, the drying process is dependent on thenonsolvent composition. A three-step drying process takes place forbeads swollen with water/ethanol mixtures with a water content higherthan 20%, while a two-step drying process is observed when the watercontent is lower than 20%. To describe the drying behavior governedby the cellulose concentration and nonsolvent composition, a simplifiedphase diagram is proposed.The drying behaviorof regenerated cellulose gel beads swollenwith different nonsolvents ( The mechanism of cellulosedissolution has been intensively studied using several different solventsystems, notably mixtures of lithium chloride and N,N-dimethylacetamide (LiCl/DMAc),18 which is used in this work. However, a limited number of studieshave investigated the kinetics of the microstructural evolution duringthe drying of regenerated cellulose.19 Thisis a crucial step in shaping cellulose materials from the wet state;it is essential to determine how the micro- or nanoscale structureschange during the removal of water or other regenerating solvents.To simplify these studies, it is important to prepare well-definedmodel systems for the cellulose-based materials. It has already beenshown that cellulose gel beads, which are smooth on the nanoscaleand can be prepared by precipitating the cellulose solution into anonsolvent (ethanol or water), can be used as a suitable model system.25 This is mostly due to the fact that it has been possible to accuratelycharacterize the highly homogeneous structure of cellulose beads andshow that they are composed of a noncrystalline, molecularly dispersedcellulose network.22 Such systems have already beenused to investigate the swelling behavior of wet, delignified cellulosicwood fibers;22 the adhesion of two cellulosesurfaces;25 and the influence of polymer grafting onthe adhesion of modified beads on a molecular scale.23 However, the structural developments during drying fromdifferent solvents and for different starting concentrations of thecellulose solution are still unexplored research areas.Celluloseis one of the mostabundant natural polymers on earth and has been used in a varietyof applications due to its excellent physical, mechanical, and biocompatibleproperties.26 For example, grazing incidence small-angle X-rayscattering (GISAXS) has been used to investigate supramolecular rearrangementsin cellulose thin films during the conversion of trimethylsilyl celluloseto cellulose via HCl vapor hydrolysis.28 GISAXS wasalso employed by Roth et al. to characterize the structure of spray-deposited nanocellulose thinfilms and water-induced structural rearrangements during drying.30 In our previous work, small-angle and wide-angle X-ray scattering(SAXS and WAXS) methods were utilized to trace the structural evolutionof 1.5 wt % regenerated cellulose gel beads (swollen in water or ethanol)during drying.24 Likewise, GISAXS in combinationwith AFM was used to trace the structural evolution of cellulose-celluloseinterfaces joining together during drying.25 All these efforts, performed to clarify the molecular interactionsat cellulose interfaces during drying, have had the common objectiveto quantify the relative importance of the different molecular interactionsthat are active between cellulose surfaces in the wet and dry state.Despite the common use of cellulose-based materials, such as paper,packaging products, hygiene materials, and high value-added nonwovenmaterials, it is surprisingly still not known which forces are holdingcellulose materials together. Vague arguments about the dominatinginfluence of hydrogen bonding have been filling the literature, anda recent review31 has summarized the currentknowledge about the importance of different types of molecular interactions.The lack of well-defined cellulose model materials has been, and stillis, an important factor behind the lack of clear model experimentswhere the influence of different interactions can be clarified. Thedevelopment of nanometer, smooth, and well-defined cellulose beadsis essential in this work since they are suitable for new, high-resolutionmeasuring techniques. The understanding of these interactions willalso be even more important in a future biobased society where water-basedprocesses and renewable materials will be necessary. Furthermore,by combining different types of forms of cellulose and also celluloseand other materials in these beads, they might also form the basefor new types of biocomposite materials.32 In the present work, the objective was to explore how the cellulosegel beads dry when different cellulose concentrations and nonsolventcompositions are used. Consequently, a comprehensive evaluation ofthe microstructural changes of cellulose gel beads clarifies how cellulosesurfaces consolidate during the drying of cellulose-rich materials.X-rayscattering is a powerful technique used to investigate themicro structure of almost every kind of material including cellulose.in situ SAXS and WAXS techniques. Additionally, the dryingbehavior of cellulose gel beads is discussed and illustrated in asimplified phase diagram.In this study, cellulose gel beads were prepared using differentcellulose concentrations and swollen by different nonsolvents: water,ethanol, and water/ethanol mixtures, as depicted in Figure S1), and the trend is consistentwith ethanol swollen beads. For 1.5 wt % cellulose gel beads swollenby different water/ethanol mixtures, the porous structure becomesslightly denser when the water content in the water/ethanol mixtureincreases . It is expected tobe caused by the large interaction between water and cellulose.The scanning electron microscopic (SEM) imagesof the internal structureof critical point dried (CPD) ethanol swollen beads are shown in 33 Moreover, apart from the size decrease, the transparency of thegel beads changes with evaporation, as shown in 2O, in Figure S3). In contrast, in the case of the ethanol swollenbeads . This is most likely due tothe multiscattering and reflection of light inside the ethanol swollenbeads, which have a tendency to keep their porous internal structureduring drying, as established in our previous work.24 For the water swollen beads, the fibrillar-like structurein the drying bead is compacted and the light scattering pores, initiallypresent during drying, are removed to such an extent that a transparentdry bead is formed. Schematic figures are provided on the right of From the side-view optical microscopeimages captured during dryingof a cellulose gel bead 1b, it caD/D0 values are the diameter of the bead(D) normalized to the initial diameter (D0). For the water swollen beads, D/D0 decreases linearly at short evaporation times decreases with the cellulose concentration after beingrelatively constant for the two lowest concentrations. After the firststage of drying, a fast linear shrinking with a higher rate, k2, occurs, indicating that the drying stageII has begun  in the drying stages I and II vs cellulose concentration for water swollen and ethanol swollen beads.For water swollen beads, with the exception of the 0.5 wt %-H2O bead, both k1 and k2 decrease as cellulose concentration increases. The ratedifference between k2 and k1 is reduced for samples with higher cellulose concentrations.For the 5 wt %-H2O beads, k2 is almost the same as k1, which meansthat the faster shrinking during drying stage II is not as pronouncedas with the lower concentration cellulose beads. In the case of ethanolswollen beads, it is observed that k1 decreasesas cellulose concentration increases. Furthermore, the values of k1 are significantly larger for the ethanol swollenbeads than the k1 values of water swollenbeads with the same cellulose concentration, which is most likelydue to the faster evaporation rate of ethanol (due to its lower vaporpressure) at ambient conditions.D/D0vs cellulose concentration for water swollenand ethanol swollen beads. It can be seen that the D/D0 value increases with higher celluloseconcentrations for both water swollen and ethanol swollen beads, indicatingthat higher cellulose concentrations better preserve the initial beadsize. Interestingly, for cellulose concentrations lower than or equalto 1.5 wt %, the equilibrated D/D0 values of ethanol swollen beads are smaller than thosefor the corresponding water swollen beads. This would suggest thatthese ethanol swollen beads shrink more than their water swollen beadcounterparts. However, when higher cellulose concentrations are used(4.0 and 5.0 wt %), the equilibrated D/D0 values determined for ethanol swollen beads are higherthan those of the corresponding water swollen beads, meaning thatthe ethanol swollen beads shrink less. This phenomenon, together withthe aforementioned slower shrinking rate (k1) for the 0.5 wt %-H2O bead, are linked to the microscalestructural evolution of beads during drying from different solvents, the SAXS curves remain relatively unchanged, and consequently,it is assumed that there is no structural change in the beads in thisregime. These three drying regimes were observed in all water swollenbeads with a cellulose concentration of 4.0 wt % or less  and corresponding “dimensionality”parameters (s1 and s2) are defined to characterize the shape of the objects presentwithin the system. The fit functions and analysis procedure are summarizedin detail in the literature36 as well as in our previouswork.24 Briefly, Rg1, in the size range of the anhydroglucoseunit, is related to the local cellulose monomer, and Rg2 corresponds to the size of the elongatedaggregate structures of the anhydroglucose units inside the gel beads.The shape of the structures can be determined by s1 and s2: when s1 = s2 = 0, the scatteringobject has a spherical symmetry; if s1 = 1 and s2 = 0, it has a cylindricalshape; and when s1 = 2 and s2 = 0, it corresponds to a lamellae structure with equalwidth and length. However, the real-world microscopic structure ofthe cellulose beads is nonideal and complex, especially during drying,we suggest that it is best represented by a combination of the aforementionedstructures. Figure S6 shows raw data measuredduring the drying of a 1.0 wt %-H2O bead and the correspondingfitting curves.To quantitatively evaluate themicroscale structural changes ofthe cellulose beads, a Guinier-Porod modelRg1, Rg2, s1, and s2 values of all the waterswollen beads throughout the drying process. As with the macroscopicdrying behavior observed in Rg1 values can be divided into three regimes (2O swollen beads (blue curve in Rg1 decreases from 0.78 to 0.71 nm during the first12 min (regime I) and then quickly increases to 1.2 nm during thenext 8 min (regime II), after which it decreases to 0.6 nm in 15 minbefore steadily decreasing to 0.5 nm (regime III). For beads withcellulose concentrations of 1.0 wt % and higher, all the water swollenbeads have similar Rg1vs time curves with three regimes. The exceptionto this is the 5.0 wt %-H2O beads on which no regime IIIwas observed due to not practically taking the measurements over asufficiently long period of time for these samples to dry to thisstage. At higher cellulose concentrations, the Rg1vs time curves are stretchedfor each regime due to the slower drying rate of these beads. Themaximum value of Rg1 decreasesfrom 1.2 to 0.9 nm when the cellulose concentration is increased from1.0 wt % to 5.0 wt %. In regime III, the Rg1 decrease is slower, with a higher value at 60 minfor higher concentration cellulose beads. For the 0.5 wt %-H2O and 0.7 wt %-H2O beads, similar trends in the curvesare observed. However, the SAXS curves are quite noisy in the high q region in the later drying phase  size in 1.0 wt %-H2O beads  measured for the 0.5 wt %-H2O bead compared with the 0.7 and 1.0 wt %-H2O beads  with drying time, illustrated in Rg1, Rg2, s1, and s2vs time  are similar to those of the 0.5 wt %-H2O bead, where a sharp increase in Rg2 is observed. This indicates that the sharp structuralchange can also occur in the ethanol swollen bead as long as the celluloseconcentration is sufficiently low, for example, 0.5 wt %.The SAXS measurements were also performed on ethanol swollen beadsas they dried, and the extracted 1D SAXS curves are shown in ng phase 6g. A simeen here 6h′. ThusD/D0 values of 0.5, 0.7, 1.0, and 1.5 wt %-H2Obeads are larger than the values of the corresponding ethanol swollenbeads. Notably, the equilibrated D/D0 values of 4.0 and 5.0 wt %-H2O beads arelower than those of 4.0 and 5.0 wt %-EtOH beads. First, there is nostructural transition in the 5.0 wt %-H2O bead  indentationexperiments,33 we know that the Young’smodulus determined in the early drying phase for the water swollenbead is lower than for an ethanol swollen bead with the same celluloseconcentration. This means that the water swollen bead has a highertendency to deform in the early drying phase. Third, the surface tensionof water is 72 mN/m, which is three-times higher than that of ethanol,22 mN/m.37 Given these three facts, itfollows logical intuition that 5.0 wt %-H2O beads are moreeasily deformed than 5.0 wt %-EtOH beads. The same is valid for the4.0 wt % beads, although the size of Rg2 for 4.0 wt %-H2O bead does increasevery slightly from 4 to 5.5 nm in the later drying phase  are assigned to scattering peaksfrom water and ethanol, respectively.43 For all thebeads, no scattering peaks from the cellulose crystalline structurewere observed before drying, which indicated that the swollen beadsare amorphous. In the early drying phase, the intensity of the scatteringpeaks from the nonsolvents (water or ethanol) decreases quickly dueto evaporation. After this, the scattering peaks from the celluloseII structure appear and their intensity increases with evaporationtime for the 0.5 wt %-H2O, 1.5 wt %-H2O, and0.5 wt %-EtOH beads. This means that the crystalline structures (celluloseII) are formed in drying phase II and grow as the drying time increases.In the SAXS results, a sharp increase of Rg2, which measures the size of the aggregate structures,was also observed for 0.5 wt %-H2O, 1.5 wt %-H2O, and 0.5 wt %-EtOH beads in drying phase II. Thus, we can correlatethese two phenomena and propose that cellulose chains reorganize intolarger crystalline aggregates, as illustrated by the gray domainsin Rg2 observed in the SAXS measurements of the same bead  suggest that cellulose-II structure was formedand distributed homogeneously inside the gel beads in the later dryingphase.To investigate whetherany crystalline order is developed during the drying process, WAXSmeasurements were conducted for 0.5 and 1.5 wt % beads swollen inwater and ethanol; the corresponding WAXS curves measured during thedrying process are summarized in ame bead 6h. This 2O bead , Rg2 only slightly increases from 2.5 to 4.5 nm meaning that theaggregation size gets roughly two-times larger. It is significantlylower than for the other water/ethanol mixtures  during the drying processare shown in 2O bead 8a. Howevm–1 from 8b–e. In m–1 from 4, it canOn the basis of the results obtained from SAXS and WAXS measurements,a simplified schematic illustration summarizing the structural evolutionof cellulose gel beads drying from water, ethanol, and water/ethanolmixtures is presented in in situ optical microscopy, SAXS, and WAXS. From the macroscale changes in bead diameter and the micro or nano scale structuralevolution during drying, a two-step or a three-step drying processis observed, depending on the cellulose concentration and the nonsolventused. A simplified phase diagram is proposed to describe the cellulosegel beads’ drying behavior. When the cellulose concentrationis lower than 0.5 wt %, a three-step drying process is observed forall the gel beads studied, regardless the ratio of water to ethanolin the nonsolvent used. A sharp structural transition correspondingto the formation of cellulose II crystalline structures occurs duringthe three-step drying. When the cellulose concentration is higherthan 5.0 wt %, a two-step drying behavior is observed, independentof the nonsolvent composition, no structural transition occurs inthese beads. The drying behavior is more complicated when the celluloseconcentration is between 0.5 and 5.0 wt %. Both two-step and three-stepdrying behaviors can be observed depending on the water content ofthe nonsolvent. For example, for 1.5 wt % gel beads, a three-stepdrying behavior occurs when the water content is higher than 20% inthe water/ethanol mixture, and a two-step drying behavior is observedwhen the water content is lower than 20%. The results presented hereinfurther our understanding of the drying behavior of cellulose on amolecular level. Such advances are invaluable for the preparationof cellulose-based materials such as fibers, membranes, and adhesives.It is also of interest that crystalline cellulose II structures canbe developed during drying when the regenerated aggregate structuresare able to reorganize at lower concentrations of the cellulose.The drying behaviorsof regenerated cellulose gel beads with differentcellulose concentrations and swollen by water, ethanol, or water/ethanolmixtures were investigated. The drying kinetics were traced by 44 Lithium chloride , N,N-dimethylacetamide , andethanol  were purchased from Sigma-Aldrich. All chemicalswere used without further purification.Domsjö dissolving pulp fibers  are the raw material used to prepare the cellulose/DMAc/LiClsolutions and gel beads. The fibers from this dissolving pulp contain96% glucose.46 The dissolving grade fibers were prewashed with deionizedwater to remove metal ions and dissolved colloidal substances . Water saturated dissolving fibers containing5.0 g of dry mass were solvent exchanged with ethanol and then DMActhrough multiple washing/filtration steps. The solvent-exchange wasperformed over 2 days for each solvent, the solvent being changedat least twice a day, using 150 mL each time. After the solvent exchange,100 mL of DMAc was heated to 105 °C for 20 min in an oil bath,and 7 g of LiCl was heated in an oven at 105 °C for 30 min toremove entrapped water. The dehydrated LiCl was added to the heatedDMAc and then allowed to cool to 65 °C at which point the DMAcsaturated pulp was added. After stirring overnight, the 5.0 wt % cellulose/LiCl/DMAcsolution was obtained. The same procedure was performed to prepare1.5 wt % cellulose/LiCl/DMAc solution. Part of 5.0 wt % solution wasdiluted to 4.0 wt % and part of 1.5 wt % solution was diluted to 1.0,0.7, and 0.5 wt % with DMAc.To makethe cellulose gel beads, cellulose/LiCl/DMAc solutions with differentcellulose concentrations were first prepared according to a previouslyestablished protocol.Figure S1. The precipitation was performedusing an infusion pump . The prepared beads were left to equilibrate for 24 h in theethanol baths. Then the beads prepared from 5.0, 4.0, 1.0, 0.7, and0.5 wt % solutions were divided into two fractions: (i) beads washedwith Milli-Q water and (ii) beads washed with ethanol. Both fractionswere washed with their respective solvents for at least 7 days toensure a proper removal of the DMAc/LiCl. Beads obtained by washingwith water or ethanol (96 vol %) are labeled water swollen beads  and ethanolswollen beads , respectively.The 6 differentconcentrated cellulose/DMAc/LiCl solutions were precipitated dropwiseinto nonsolvent baths , where the cellulose solutiondrop solidified into the initial cellulose gel beads, as depictedin 2O, 1.5 wt %-H2O/EtOH(80/20), 1.5 wt %-H2O/EtOH(60/40), 1.5 wt %-H2O/EtOH(40/60), 1.5 wt %-H2O/EtOH(20/80), and 1.5wt %-EtOH.For the beads preparedfrom the 1.5 wt % solution, they were divided into six fractions andwashed with Milli-Q water, Milli-Q water/ethanol mixtures  or ethanol for at least 7 days.These were labeled as 1.5 wt %-H47 The ethanol swollen cellulosebeads were solvent exchanged to pure ethanol over 2 days refreshingthe solvent three times per day. The beads were then placed in theCPD chamber  and liquid carbon dioxidewas injected into the chamber under a pressure of ∼50 bar forsolvent exchange from ethanol to CO2. The conditions ofthe chamber were then brought above the CO2 critical point,to ∼100 bar and 36 °C, after which the chamber was depressurizedand the CO2 evaporated.To observe the morphologyof the initial cellulose gel beads with a scanning electron microscope(SEM), ethanol swollen beads were chosen to prepare the dry samplesusing a critical point drying method (CPD), during which the capillaryforces between the vapor, liquid, and solid cellulose is theoreticallyexcluded.The interior morphologies ofthe CPD dried cellulose beads were characterizedusing a S-4800 field emission scanning electron microscope (FE-SEM) operating at high vacuum. CPD dried beadswere cut and glued onto a conductive carbon tape on the sample holderand then coated with Pt/Pd in a Cressington 208 HR sputter coater for 20 s to limitsample charging during imaging.An optical camera  was used to monitor the diameter of thecellulose gel beads throughout the drying process at 26 °C and33% RH (the same conditions used in the SAXS/WAXS measurements). Tobe consistent with SAXS/WAXS measurements, Kapton tape (part number:42–020–0016) was used as the substrate for this characterizationas well.In situ SAXS/WAXS characterization for drying cellulosegel beads was performed at Forschungszentrum Jülich, Germany.The X-ray source is a D2-MetalJet (Excillum) with a liquid metal anodeoperating at 70 kV and 3.57 mA with Ga–Kα radiation (wavelengthλ = 0.1314 nm), providing a brilliant and narrow beam (<100μm). The X-ray beam was further focused with a focal lengthof 55 cm, using a specially made X-ray optic (Xenocs) to provide avery narrow (0.15 × 0.15 mm2) and intense beam atthe sample position. The scattering data were acquired with a position-sensitivedetector  with a pixel size of 172 μm.After calibration with silver behenate, the sample-to-detector distanceswere set to 1107 mm and 152 mm for SAXS and WAXS measurements, respectively.The cellulose beads were adhered to the Kapton tape surface, whichprevents the beads from sliding when the sample holder was placedin the vertical position. Individual 2D scattering patterns were recordedfor water and ethanol swollen beads. The SAXS scans took 30 s eachand the WAXS took 15 s to scan. After radial integration, the backgroundscattering of the Kapton tape was scaled and subtracted for each curveto obtain more accurate data. Scaling was completed to account forthe change in X-ray transmission that occurs from bead shrinking duringdrying.