This study aimed to develop a drug delivery system with hybrid biodegradable antifungal and antibacterial agents incorporated into poly lactic-co-glycolic acid (PLGA) nanofibers, facilitating an extended release of fluconazole, vancomycin, and ceftazidime to treat polymicrobial osteomyelitis. The nanofibers were assessed using scanning electron microscopy, tensile testing, water contact angle analysis, differential scanning calorimetry, and Fourier-transform infrared spectroscopy. The in vitro release of the antimicrobial agents was assessed using an elution method and a high-performance liquid chromatography assay. The in vivo elution pattern of nanofibrous mats was assessed using a rat femoral model. The experimental results demonstrated that the antimicrobial agent-loaded nanofibers released high levels of fluconazole, vancomycin, and ceftazidime for 30 and 56 days in vitro and in vivo, respectively. Histological assays revealed no notable tissue inflammation. Therefore, hybrid biodegradable PLGA nanofibers with a sustainable release of antifungal and antibacterial agents may be employed for the treatment of polymicrobial osteomyelitis. Candida albicans and Staphylococcus aureus are the nosocomial pathogens frequently responsible for severe morbidity and mortality, even with appropriate treatment. Coinfection is associated with mixed biofilms, more severe clinical manifestations, and enhanced drug resistance [Despite advances in surgery, chronic osteomyelitis treatment remains a great challenge, often associated with a significant financial burden on healthcare systems. Osteomyelitis is an acute or chronic inflammatory process affecting the bone and its structure, secondary to the infection with pyogenic organisms, including bacteria, fungi, and mycobacteria ,2. Certasistance ,7,8. Thesistance ,7. Antifsistance ,10.The consensus treatment for bacterial and fungal osteomyelitis often involves both surgery and administration of antimicrobial agents ,12. AntiS. aureus osteomyelitis [In this study, we developed hybrid biodegradable antifungal and antibacterial poly lactic-co-glycolic acid (PLGA) nanofibers via electrospinning that provide extended release of fluconazole, vancomycin, and ceftazidime. Fluconazole is an antifungal agent used against several fungal infections, including candidiasis, blastomycosis, coccidioidomycosis, etc. . Vancomymyelitis . Ceftazimyelitis . Electromyelitis . Electromyelitis . Owing tmyelitis ,24,25,26myelitis ,28.Our aim was to manufacture and assess electrospun antimicrobial-loaded degradable PLGA nanofibers and nanofibrous mats, and determine their in vitro and in vivo antimicrobial agent discharge pattern as a possible treatment for osteomyelitis.Nanofibers incorporated with biodegradable hybrid antimicrobial agents were successfully manufactured via electrospinning. The wetting angles of spun mats decreased with the increasing percentage of pharmaceuticals, that is, 125.6°, 107.1°, and 117.8°, for the pristine PLGA nanofibrous mats, fluconazole-, and vancomycin/ceftazidime-embedded mats, respectively . The flu−1 was promoted by the C–H bonds of vancomycin and ceftazidime [−1 was caused by the –NH bond of the added pharmaceuticals [−1 were significantly developed for pharmaceutical-loaded nanofibers with respect to the peak of the pristine PLGA nanofibrous mats. The FTIR assay results confirmed the successful incorporation of antimicrobial agents into the electrospun hybrid nanofibers  for up to 56 days in vivo .Over the past three decades, the pathogenesis of osteomyelitis has been clarified, along with the identification of factors responsible for infection. In addition to surgical treatment, many antimicrobial agents have been adopted for the treatment of osteomyelitis. Bacterial and fungal coinfection has been reported in upper respiratory tract infections and cultures from distinct medical devices, including dentures, implants, endotracheal tubes, and, most commonly, catheters . AlthougCandida albicans and Staphylococcus, lead to an increase in antimicrobial agent resistance by a hundred-fold of MIC [S. aureus and C. albicans coinfection requires serial and radical debridement and the use of a higher dose of fluconazole (800 mg/day or 12 mg/kg body weight) than a regular dose of 400 mg/day for an extended period. However, the role of antimicrobial agent-loaded bone cement beads was not clearly described in their study [The major difficulty in the treatment of osteomyelitis or other co-infections is biofilm formation. Three-dimensional bacterial and fungal biofilms protect the microbial community from antimicrobial agent damage, increasing chronic infections . This dod of MIC ,10. As mir study ,36,37,38ir study ,40, inclir study .Advances in nanotechnology have led to encouraging treatments for osteomyelitis. In this study, degradable PLGA nanofibers were exploited for the sustained discharge of antimicrobial agents into the target area for infection control. Due to their elevated surface-area-to-volume ratio, nanofibers offer an advantageous vehicle for the transport of water-insoluble or poorly soluble pharmaceuticals. The three-dimensional network structure of nanofibers also mimics the architecture of the extracellular matrix of natural tissues, allowing enhanced cell functionality after the incorporation of drugs and multiple factors . Our preSeveral factors may affect the release of pharmaceuticals from PLGA matrix systems , includiThe advantage of local drug delivery is high, sustained, local levels of pharmaceuticals without high systemic doses, thus, minimizing systemic toxicity . IntraveTraditionally, osteomyelitis is treated with 4–6 weeks of parenteral antimicrobial agents after debridement procedure ,13,52, aDespite the proven effectiveness of the hybrid antimicrobial agent-incorporated nanofibrous mats, there were limitations in this study. First, restriction lies in the limited type of animals used. Second, a non-infected animal model was used in this study. It is unknown whether the hybrid drug-eluting PLGA nanofibers will perform differently in an infected model. Finally, the relationship between these findings and osteomyelitis in humans is unclear and requires further investigation.PLGA was used in nanofiber fabrication . Vancomycin hydrochloride, ceftazidime hydrate, fluconazole, and hexafluoro-2-propanol (HFIP) (Sigma-Aldrich) were used as the antimicrobial agents and solvent, respectively.Two-layer hybrid nanofibers were prepared using a lab-scale electrospinning apparatus. To fabricate the antifungal-loaded nanofibers, 1120 mg PLGA and 560 mg fluconazole were blended with 6 mL HFIP and subsequently spun into nanofibrous mats using a syringe/needle  at a temperature of 25 °C and relative humidity of 65%. The mixture transport speed was 0.5 mL/h. The voltage employed was 18,000 V, and the distance between the needle and the collector was 150 mm. This was followed by electrospinning of the PLGA/vancomycin/ceftazidime nanofibers. PLGA/vancomycin/ceftazidime (1120 mg/280 mg/280 mg) was blended with 6 mL of HFIP using the same spinning parameters as those for PLGA/fluconazole nanofibers. After electrospinning, hybrid antifungal and antibacterial nanofibers were obtained, with a thickness of approximately 0.18 mm. For comparison, pure PLGA nanofibers were also prepared by mixing PLGA (1120 mg) with HFIP (6 mL) and electrospinning into nanofibrous mats.The electrospun nanofibrous mats were evaluated using a Joel JSM-7500F scanning electron microscope . One hundred randomly selected nanofibers from the SEM micro-image were used to determine the distribution of the nanofiber size.A general-purpose goniometer  (N = 3) was used to evaluate the wetting angle of the nanofibrous mats.The tensile properties of the nanofibers were evaluated using a Lloyd tensiometer . Nanofibrous samples with dimensions of 10 mm × 50 mm were clamped between two grips with a distance of 3 cm between the grips. The sample was stretched by the top grip at a rate of 60 mm/min for a distance of 10 cm (N = 3) before the grip returned to its starting point.The thermal properties of pristine PLGA, vancomycin/ceftazidime-loaded PLGA, and fluconazole-loaded PLGA were identified using TA-DSC25 differential scanning calorimeter . The heating rate was maintained at 10 °C/min over a 30–350 °C scan range.−1 with 32 scans, with a 400–4000 cm−1 range.The spectra of the electrospun antimicrobial agent-incorporated mats were obtained using a Thermo Fisher Nicolet iS5 Fourier-transform infrared (FTIR) spectrometer . The nanofibrous samples were pressed into KBr discs and evaluated under the absorption mode. The resolution was 4 cmm is the amount of drug measured in the nanofibrous mat, and Wa is the drug added in the electrospun fibers.To determine the entrapment efficiency (EE%) of added drugs, the nanofibrous mats with vancomycin/ceftazidime or fluconazole were weighed accurately in triplicate and extracted in HFIP employing a magnetic stirrer at 20 rpm. The extract solution was then centrifuged at 10,000 rpm for 10 min at ambient temperature. The drug concentrations in the eluents were characterized using a Hitachi L-2200R high-performance liquid chromatograph  (N = 3). The EE % was calculated by the following equation :(1)EE(%)The discharge pattern of antimicrobial agents from drug-incorporated nanofibrous mats was assessed using an in vitro elution scheme. Nanofibrous mats with a dimension of 10 mm × 10 mm (~5 mg) were submerged in a phosphate-buffered solution of 1 mL at 37 °C. The solution was then collected and evaluated after 24 h of isothermal incubation. The buffer was refreshed every 24 h, and the process was repeated for 30 days. The concentrations of fluconazole, vancomycin, and ceftazidime in the collected solutions were determined using the HPLC assay.All animal-correlated processes received institutional approval (CGU109-005), and all animals were cared for under the supervision of a licensed veterinarian, in line with the ARRIVE guidelines and the regulations of the Department of Health and Welfare, Taiwan.Male Sprague Dawley rats  were enrolled in the tests. The rats were kept in independent cages with temperature and light control and had ad libitum access to standard mouse chow and sterilized drinking water.The rats first received general anesthesia through isoflurane inhalation provided by a vaporizer in a polymethyl methacrylate box. Anesthesia was maintained through inhalation of isoflurane via a mask. After sedation, the right thigh of each rat was depilated, cleaned using soap, and disinfected with 70% ethanol prior to surgery. The right femoral shaft was explored using an anterolateral approach under aseptic conditions. Membranes (1 cm × 2 cm) were cut from the electrospun fluconazole/vancomycin/ceftazidime-loaded nanofibers a and surWe prepared degradable antimicrobial agents incorporated in PLGA nanofibers and assessed their release behaviors. The in vitro drug discharge was assessed using HPLC, whereas a rat bone model was used for the evaluation of in vivo drug elution. The results showed that all nanofibrous mats discharged effective levels of vancomycin, ceftazidime, and fluconazole in vitro for 30 days. Animal tests also showed that the nanofibers released effective concentrations of antimicrobial agents for over 56 days after surgery. Histological assays did not reveal any notable tissue inflammation. Therefore, degradable nanofibers with sustained release of antimicrobial agents may be a potential treatment for polymicrobial osteomyelitis.


In this study, a simple and fast detection method of Hg2+ based on the molecular beacon aptamer was established, according to the principle that Hg2+ could change the structure of the molecular beacon aptamer, resulting in the changed fluorescence intensity. All of the detection conditions were optimized. It was found that an optimal molecular beacon aptamer MB3 showed the optimal response signal in the optimized reaction environment, which was 0.08 μmol/L MB3, 50 mmol/L tris buffer , and a 10 min reaction. Under the optimal detection conditions, the molecular beacon aptamer sensor showed a linear response to Hg2+ concentration within a range from 0.4 to 10 μmol/L and with a detection limit of 0.2254 μmol/L and a precision of 4.9%. The recovery rates of Hg2+ in water samples ranged from 95.00% to 99.25%. The method was convenient and rapid, which could realize the rapid detection of mercury ions in water samples.Biosensors for mercury (II) (Hg Low dose and long-term exposure will cause chronic damage to human organs, such as the liver, kidney, reproductive system and nervous system [2+, including atomic absorption spectroscopy (AAS) [2+. It is important to develop rapid, convenient and cost-efficient detection methods for Hg2+.Mercury is one of the most toxic heavy metals with well documented impacts to the environment and human health ,2, with s system . In recepy (AAS) ,5, atomipy (AAS) , inductipy (AAS) , surfacepy (AAS) , etc. Ho2+, the T-rich oligonucleotide has been frequently applied as a Hg2+ specific aptamer (MSA) [Aptamers are oligonucleotides that can specifically bind to proteins or other small molecules with high affinity and specificity. They are selected using an in vitro selection procedure called SELEX, the systematic evolution of ligands with exponential enrichment ,10. Afteer (MSA) ,12.A molecular beacon is a stem-ring double-labeled oligonucleotide probe that forms a hairpin structure at the 5′and 3′ ends. The loop region contains an antisense recognition domain against a nucleic acid target, and the absence of a target allows the molecular beacon to self-hybridize at the stem . Since tGenerally, a molecular beacon probe contains 25–35 nucleotides, and its structure includes loop region, stem region and labeling region. The loop region is composed of 15 to 30 nucleotides, which can specifically bind to the target molecule. The stem region is composed of five to eight base pairs, which can be reversibly dissociated when the molecular beacon binds to the target molecule. In the labeling region, the fluorescent group is generally labeled at the 5′ end, and the quenching group is generally labeled at the 3′ end. In the free state, the molecular beacon has a hairpin structure, and the fluorescence groups and quenching groups are close to each other; at this time, the fluorescence resonance energy transfer occurs, and the fluorescence is almost completely quenched. When the molecular beacon binds to the target molecule, its spatial configuration changes, the distance between the fluorescence group and the quenching group increases, and the fluorescence of the molecular beacon is restored. Compared with other types of nucleic acid probes (such as constructing DNA double-stranded hybridization system), the structure of the molecular beacon is simple, and one kind of molecular beacon can detect one kind of target in a homogeneous system. At the same time, it has the characteristics of low background fluorescence, simple operation, high sensitivity and strong specificity.2+ detection. The fluorescent group was labeled at the 5′ end and the quenching group was labeled at the 3′ end. When Hg2+ was added, the T base in the molecular beacon aptamers formed the structure of T-Hg2+-T. The specific binding of T base and Hg2+ led to the conformational change in the molecular beacon aptamer, which enlarged the distance between the fluorescence group and quenching group, so the fluorescence intensity increased. Based on this principle, a rapid detection method for Hg2+ was established, which developed a new way for the detection of Hg2+.A molecular beacon aptamer combines the advantages of an aptamer and molecular beacon organically. In this paper, a molecular beacon aptamer was constructed and used for HgA FilterMax F3 multi-mode microplate reader , 96-well, black microplate , PHS-3B pH meter  and a TGL-16 high-speed refrigerated centrifuge  were used.2, HCl, HgCl2, ZnCl2, AlCl3, MnCl2, SnCl2, CoCl2, KCl, CuCl2, BaCl2, CaCl2 and CdCl2 were used as the analytical reagents. The water used in the experiment was ultra-pure water.Tris, NaCl, MgClThe DNA sequences used in the experiment were all synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd. and the following DNA sequences were used: MB1, GAACACCCTTCTTCTTCCTTGTTGTTC ; MB2, GAACACCCCTTC TTCTTCCTTGTTGTTC ; MB3, GAACACCCCCTTCTTCTTCCTTGT TGTTC .2, pH 7.2), 50 μL of 0.1 μmol/L molecular beacon aptamer was added to 50 μL of 50 μmol/L Hg2+ solution (the sample group) or 50 μL buffer (the blank group). After they were mixed, the fluorescence intensity was detected every five minutes by a multi-function microwell plate detector in a black 96-well plate, with an excitation wavelength 485 nm and emission wavelength of 535 nm.In the buffer  or 50 μL buffer (the blank group). Afterwards, the fluorescence intensity was detected according to the method in I and 0I were the fluorescence intensity of the sample group and the blank group, respectively, and the fluorescence intensity change ∆I (∆I =0 I − I) was calculated.The MB3 with the best sensitivity to Hg2 concentration  in the buffer were studied. Under the optimal reaction and test conditions, the fluorescence intensity was detected and the ∆I was calculated according to the method in After the optimal molecular beacon aptamer concentration and the optimum reaction time were determined, the effects of different pH , NaCl concentration  and MgCl2+, Al3+, Mn2+, Sn2+, Co2+, K+, Cu2+, Ba2+, Ca2+, Cd2+ and Hg2+ in the optimized buffer solution were added. The detection and calculated methods were the same as In the blank group, 50 μL of optimum concentration MB3 and 50 μL of optimized buffer solution were added. In the sample group, 50 μL of optimum concentration MB3 and 50 μL of 50 μmol/L Zn2+ at different concentrations were added. The ∆I was calculated, and the relationship between the concentration of Hg2+ and the ∆I was established. The Kd of MB3 was fitted and calculated by GraphPad Prism 5 software.The blank group experiment was the same as According to the Operational Guidelines for Water Sample Detection of Global Environmental Monitoring System, when the confidence level was 95%, the detection limit was the one-time measured value of the sample concentration, which was significantly different from the one-time measured value of the zero concentration sample. When the number of blank measurements was greater than 20, the detection limit was 4.6 times that of the blank standard deviation. The precision of the method was evaluated by the relative standard deviation of five repeated tests.2+ was detected. The 0.8, 4 and 8 μmol/L Hg2+ solutions were added to the filtered water samples. Then, the samples were tested according to the operation of Water from tap water was collected and filtered with a 0.22 μm filter membrane, and the initial concentration of HgP was the recovery rate, C1 was the initial concentration of Hg2+ in the water, C2 was the determination of Hg2+ concentration in the standard addition group, and C3 was the standard adding concentration of Hg2+.2+ was added, the T-base and Hg2+ formed the structure of T-Hg2+-T. This led to the changed structure of the molecular beacon aptamer, the distance between the fluorescent group and quenching group became longer, and the fluorescence intensity increased greatly. The concentration of Hg2+ was directly proportional to the change in fluorescence intensity of the molecular beacon aptamer, so the concentration of Hg2+ could be detected.As shown in Three molecular beacon aptamers, MB1, MB2 and MB3, were designed in the experiment, and the MFold webserver was used to predict their secondary structures. As shown in 2+. The results were shown in 2+, the fluorescence intensities increased to 22,549.000, 26,630.130, and 28,116.130, respectively, and the fluorescence intensity after adding Hg2+ was 2 to 4 times higher than that without Hg2+.The experiment investigated whether the molecular beacon aptamer can be successfully closed and opened by detecting the fluorescence intensity of the system before and after adding Hg2+ smoothly. Among them, the fluorescence intensity change in MB3 was the highest, so MB3 was chosen as the optimal molecular beacon aptamer.This proved that three molecular beacon aptamers were able to bind to Hg2+ were detected. As can be observed from I was increased, and it was the highest at 0.08 μmol/L. When the concentration of MB3 was further increased, the change value of fluorescence intensity was decreased. When MB3 concentration was 0.08 μmol/L, with the extension of reaction time, ΔI showed a slight increase at first and then gradually decreased, and reached the maximum value at 10 min.The changes in fluorescence intensity of the different concentrations of MB3 reacting with 50 μmol/L Hg2+ was 50 μmol/L, the concentration of MB3 was 0.08 μmol/L, and the incubation time was 10 min, the effects of buffer pH  on the detection system were studied. The results  on the fluorescence intensity of the detection system were studied. The results ; the results were shown in 2 reached 10 mmol/L, the ΔI was the highest.Under the conditions that the concentration of Hg results B showed I of MB3 interacting with different metal ions was detected, and the results are shown in ΔI of MB3 reacting with Hg2+ was the highest (ΔI = 47161.375), while those of other metal ions were relatively low or even had no change. Among the other ten kinds of metal ions, Ba2+, Ca2+, Cd2+, Al3+ and Co2+ had a relatively large influence on the detection, and the ΔI was 2732.875, 1834.500, 1546.125, −1356.500 and −780.200, respectively. However, their ΔI accounted for less than 5.79% of the ΔI of Hg2+. It was proved that the molecular beacon aptamer MB3 had excellent specificity.Under the optimal reaction conditions, ΔI of MB3 reacting with 0.4–50 μmol/L Hg2+ was detected, and the results are shown in 2+ concentration from 0.4 to 10 μmol/L, the change in fluorescence intensity increased with the increase in Hg2+ concentration. However, when the Hg2+ was higher than 10 μmol/L, ΔI tended to be stable. The dK of MB3 was fitted and calculated by GraphPad Prism 5 software. It can be observed from dK of the MB3 was 8.567 ± 1.558 μmol/L.Under the optimal conditions, ΔI had a linear relationship with the concentration of Hg2+. The standard curve was y = 3506.6x − 960.57; the linear regression coefficient was 0.9901 -mediated DNA duplexes [2+ analysis. The constructed aptamer biosensors for Hg2+ detection include colorimetric sensors [Aptamers have received more and more attention in the construction of biosensors in the past few years. Recently, various strategies based on aptamers for metal ion detection have been developed, including Hg2+ ,17, Pb2+2+ [2+ , Cd2+ [1 [2+ [2+  and so oduplexes . Based o sensors ,20, fluo sensors ,22, elec sensors ,23 and s sensors ,24,25, a2+ detection was designed. In the detection system, there was only one type of nucleic acid probe, and there was no need to hybridize various types of the nucleic acid probe, so the detection system and operation are relatively simple. The results showed that Hg2+ could induce the conformational change in the molecular beacon aptamer by forming a T-Hg2+-T structure, resulting in the fluorescence signal changes to realize the detection of Hg2+. The MB3 probe designed in this study had strong affinity, and the dK of the MB3 was 8.567 ± 1.558 μmol/L. The study of Qu. et al. [dK of the selected aptamer binding to Hg2+ was 1.49 ± 1.558 μmol/L, and the dK of the previously reported Hg2+ aptamer [Kd of the MB3 probe was between 1.49 ± 1.558 μmol/L and 34.08 ± 4.07 μmol/L. This could also prove that the designed molecular beacon was successful and effective.In this study, a simple and convenient “signal on” fluorescence sensor was constructed. The advantages of an aptamer and molecular beacon were organically combined, and the molecular beacon aptamer for Hg. et al.  found th aptamer  was 34.02+, Ca2+, Cd2+, Al3+ and Co2+ had a certain influence on the detection, but the overall influence was small. When the method was used for detection, the relative standard deviation of five repeated tests was 5.9%, which indicates that the precision of the method is good. At the same time, it was found that the fluorescence intensity of the reaction system changed very little and the detection was stable when the reaction time was from 10 to 35 min. The detection limit of the method was 0.2254 μmol/L.In addition, this method has good specificity, precision and stability. Among the other 10 kinds of metal ions tested, Ba2+ was demonstrated with unmodified gold nanoparticles as probes and thrombin-binding aptamer as sensing elements; the limit of detection was 200 nmol/L. This result was similar to that of our study. However, in the study of Ono et al. [2+; the limit of detection was 40 nmol/L. The detection limit is lower than ours, so our detection sensitivity needs to be improved and optimized.In the study of Wang et al. , a coloro et al. , a “sign2+, but the background value of MB3 fluorescence intensity also increased, resulting in the decreased ΔI. In terms of the reaction time, the reaction speed was very fast after adding Hg2+, and the fluorescence intensity tended to be stable at about 10 min, which laid the foundation for rapid detection. From pH and ion strength of the buffer, the weak alkaline condition and a certain concentration of sodium ion and magnesium ion were beneficial to the binding of the molecular beacon aptamer to Hg2+ and detection. Especially, the pH of the buffer system had a great influence on the results; the optimized pH in this study was 8.1, which was close to the pH of buffer system reported by previous studies. For example, Li et al. [In addition, the reaction conditions of the experiment, including the concentration of MB3, reaction time, pH and ionic strength of the buffer, were comprehensively optimized. Judging from the results, the higher concentration of MB3 was not the best. A higher concentration increased the probability of MB3 binding to Hgi et al.  used a bi et al.  used an 2+, but its detection sensitivity needs to be improved and optimized. The study focuses on the design of a nucleic acid probe and the establishment of a detection method, which is only preliminarily applied in tap water. However, its application in other samples needs to be further studied.Overall, the method has the advantages of fewer reagents, simple operation, rapid detection and low cost. It can complete the detection of mercury ions in water samples within 20 min, showing good specificity and certain sensitivity. It can be used as a rapid screening method for Hg2+ based on a molecular beacon aptamer was developed, which was used for the detection of water samples. The designed molecular beacon aptamer could specifically and closely bind to Hg2+ to form a thymine-Hg2+-thymine (T-Hg2+-T)-mediated structure, which changed the hairpin structure of the original molecular beacon aptamer and increased the fluorescence intensity of the system. The MB3 probe designed in this study had high affinity and specificity. This method had a good linear relationship in the range of 0.4–10 μmol/L. The method had a certain anti-interference ability, and the recovery rate in the water sample was over 95.00%. The method did not require a complicated sample pretreatment process, the detection system was simple, and the detection could be completed within 20 min, which paves the way for the rapid screening of Hg2+.A simple and rapid “signal on” fluorescent sensor for detecting Hg


Globally, antimicrobial resistance (AMR) and climate change (CC) are two of the top health emergencies, and can be considered as two interlinked public health priorities. The complex commonalities between AMR and CC should be deeply investigated in a One Health perspective. Here, we provided an overview of the current knowledge about the relationship between AMR and CC. Overall, the studies included pointed out the need for applying a systemic approach to planetary health. Firstly, CC increasingly brings humans and animals into contact, leading to outbreaks of zoonotic and vector-borne diseases with pandemic potential. Although it is well-established that antimicrobial use in human, animal and environmental sectors is one of the main drivers of AMR, the COVID-19 pandemic is exacerbating the current scenario, by influencing the use of antibiotics, personal protective equipment, and biocides. This also results in higher concentrations of contaminants  in natural water bodies, which cannot be completely removed from wastewater treatment plants, and which could sustain the AMR spread. Our overview underlined the lack of studies on the direct relationship between AMR and CC, and encouraged further research to investigate the multiple aspects involved, and its effect on human health. Antimicrobial resistance (AMR) is one of the most pressing health challenges globally, accounting for 670,000 annual infections in the European Union and nearly 33,000 related deaths . While aThe resulting pandemic has only further underlined the need for antimicrobial stewardship to prevent AMR . In factEnterobacteriaceae carbapenem-resistant and methicillin-resistant Staphylococcus aureus (MRSA)—can be rapid, with severe consequences for vulnerable patients [The high consumption of antibiotics in the community, and in all healthcare settings, is undeniably the main factor contributing to AMR, especially in high-income countries . This evpatients . In linepatients .Globally, AMR is associated with longer state of illness, higher mortality rates, increased costs, and ineffective antibiotic treatments. In fact, patients with AMR infections show longer hospitalization and higher treatment costs than those infected by drug-susceptible strains ,44. The In this scenario, several comprehensive strategies should be implemented to control AMR. Surveillance of antibiotic use, detection of resistance in human beings and animals, awareness campaigns on the correct use of antibiotic, and antibiotic stewardship programs in healthcare settings are just some of the interventions that countries should address ,48. CoopWhat is described in this paragraph about AMR points out similarities with CC ; for thiAccording to the WHO, AMR is one of the top 10 global public health threats facing humanity, with misuse and overuse of antimicrobials as the main drivers for the development of drug-resistant pathogens. Although AMR is a natural phenomenon, the main drivers of its development and spread are “man-made”, including misuse and overuse of antimicrobials in humans, animals and plants . FindingCampylobacter, Salmonella and Vibrio cholerae—to warmer temperatures have led to the re-emergence of these infectious diseases with the increasing temperatures in water systems [Due to its broad scale, CC has been often compared to AMR; but these two issues are even more intertwined, since CC could indirectly contribute to AMR . For thi systems ,59. Thus systems ,60,61,62 systems . Therefo systems ,64. For  systems .In the same way, intersectoral strategies may be effective also against the climate crisis, even if further efforts are still needed to understand how the One Health approach may be enough to manage CC . The majAs stated above, the effects of CC on human health is getting worse with time, as well as its impact on Public Health that affects all the physical, natural, social and behavioral dimensions . This woWhile temperatures rise as a consequence of the climate crisis, AMR is increasing in humans, animals, plants and the environment ,73. IncrAn interesting question is whether CC could explain—at least partially—differences in AMR proportions observed in Europe, a region characterized by countries with different healthcare systems. In general, increasing temperature and population density led to increased rates of AMR ,101. TheIt has also been demonstrated that warmer climates might affect heavy metals or biocides concentrations in soil and water, as well as their uptake by bacteria, triggering AMR by co-resistance mechanisms ,106,107.Rising temperatures are closely linked with flooding, population displacement and overcrowding caused by storms and precipitations . These eIn addition, extreme weather events could lead to drought that, along with food scarcity and decline in the healthcare system, determine higher risk of acquiring antibiotic-resistant enteric pathogens . InteresStorms and precipitations can also damage wastewater and sewage infrastructure, increasing the risks of floodwater pollution . NotablyIn this scenario, microplastics are defined as conventional plastic with dimension <5 mm, accumulated in landfills or in the natural environment, that fragment rather than decompose. In particular, their accumulation has been widely reported in waters, soils and air . MicroplE. coli resistant to fluoroquinolones originates from the hospital, but also from the aquatic, environment. This represents a major issue for both human and animal health, as well as a concern for the environment. With respect to the relationship between AMR and CC, antibiotic-related modifications in bacterial cells are similar to those caused by increased temperatures [It is therefore plausible to state that the impacts of CC will result in an increased use of antimicrobials in humans, animals and plants. As a result of heat stress, for instance, poultries are overtreated with antibiotics, affecting their egg production and growth rates . Globalleratures . The AMReratures . Since feratures ,151.Current evidence about the effects of CC on AMR should be translated into a common vocabulary and a fruitful dialogue between political leaders, policymakers, media, and the general public. With respect to the public, climate activists are used to shock people by painting the problem as an imminent catastrophic event. However, it is unlikely that this kind of approach could sustain behavioral changes. Instead, people should be guided in the fight against AMR and CC by reducing the prescriptions of unnecessary antibiotics . This isThe psychological distance represents a key barrier to people’s engagement with global problems, leading to a wrong and sweetened perception of the consequences on human health. In the era of expanding essential access, reducing inappropriate antibiotic use is a difficult challenge. To encourage people to act on climate crisis or antibiotic resistance, policymakers need to develop novel ways to make the issue closer to reality . A plausCountries and health systems continue to deal with the health and socio-economic effects of the COVID-19 pandemic. The economic losses associated with climate crisis determine increased pressure on economies already modified by synergistic effects of the COVID-19 pandemic . For insAs stated above, the contamination of waters with the so-called “emerging contaminants”   was a prCC and AMR are two of the greatest threats currently facing the world. Both have been exacerbated by, and can be mitigated with, human actions. In the era of climate crisis, the burden of infectious disease could be still reduced focusing on One Health strategies, recognizing how human health is closely connected to animals and the environment . As repoThe complex commonalities between AMR and CC should be deeply investigated in a One Health perspective. Our overview on the current knowledge about the relationship between AMR and CC pointed out the need for further research to deeply investigate these two intertwined global challenges. To do that, it should be clear that CC increasingly brings humans and animals into contact, leading to outbreaks of zoonotic and vector-borne diseases with pandemic potential. Moreover, it is well-established that antimicrobial use in human, animal and environmental sectors is one of the main drivers of AMR. The COVID-19 pandemic is exacerbating the current scenario, by influencing the use of antibiotics, personal protective equipment and biocides, also resulting in higher concentrations of contaminants in natural water bodies. For this reason, contaminants  cannot be completely removed from wastewater treatment plants, sustaining the AMR spread. Thus, a planetary health approach could offer potential solutions to commonly tackle these important public health issues.


N719. SFA-5–8 are synthesized and utilized as sensitizers for dye-sensitized solar cell (DSSC) applications. The effects of the donor unit (triphenylamine), π-conjugation bridge (thiophene ring), and various acceptors (phenylacetonitrile and 2-cyanoacetamide derivatives) were investigated. Moreover, this was asserted by profound calculations of HOMO  and LUMO  energy levels, the molecular electrostatic potential (MEP), and natural bond orbital (NBO) that had been studied for the TPA-sensitizers. Theoretical density functional theory (DFT) was performed to study the distribution of electron density between donor and acceptor moieties. The sensitization by the absorption of sensitizers SFA-5–8 leads to an obvious enhancement in the visible light absorption (300–750 nm) as well as a higher photovoltaic efficiency in the range of (5.53–7.56%). Under optimized conditions, SFA-7 showed outstanding sensitization of nanocrystalline TiO2, resulting in enhancing the visible light absorption and upgrading the power conversion efficiency (PCE) to approximately 7.56% over that reported for the N719 (7.29%). Remarkably, SFA-7 outperformed N719 by 4% in the total conversion efficiency. Significantly, the superior performance of SFA-7 could be mainly ascribed to the higher short-circuit photocurrents (Jsc) in parallel with larger open-circuit voltages (Voc) and more importantly, the presence of different anchoring moieties that could enhance the ability to fill the gaps on the surface of the TiO2 semiconductor. That could be largely reflected in the overall enhancement in the device efficiency. Moreover, the theoretical electronic and photovoltaic properties of all studied sensitizers have been compared with experimental results. All the 2-cyanoacrylamide derivative sensitizers demonstrated robust photovoltaic performance.Herein, we present a thorough photovoltaic investigation of four triphenylamine organic sensitizers with D–π–A configurations and compare their photovoltaic performances to the conventional ruthenium-based sensitizer  In 1991, O'Regan and Graztel described a novel type of sensitizer known as DSSCs2. They have become promising alternatives to traditional silicon-based photovoltaics because they are cheap, work well, and are easy to make3. New attempts have been proposed to present new approaches for the development of dye sensitizers. Most of such dyes are based on metal–organic complexes like ruthenium (Ru) dye sensitizers that are widely used due to their wide optical absorption, high photostability, and energy compatibility with the TiO2 layer. This in turn could achieve high DSSC performance associated with various anchoring carboxylate groups via the metal-to-ligand charge transfer (MLCT) processes4. Despite the high efficiencies of the sensitized devices with ruthenium-based dyes such as N3, N719, black dye, and HD-2, they pose many issues in terms of their high cost, scarcity of metal, and highly purified methods7. In this regard, metal-free organic sensitizers could be a good choice. There are various types of metal-free sensitizers such as carbazole, phenothiazine, coumarin, thiophene, triphenylamine, and boron dipyrromethene (BODIPY) with classifications D–π–A, D–D–π–A, and D–π–A–A. In comparison to their metal-based counterparts8, such as porphyrin dye9 and chlorophyll-based dyes10, they all have a high molar extinction coefficient, low cost, and ease of purification. Metal-free sensitizers have been widely recognized to have a complementary absorption profile with a high molar extinction coefficient and higher efficiencies12. Amongst donor units, triphenylamine, phenothiazine, carbazole, and coumarin have been commonly utilized as promising metal-free sensitizers13. In this context, TPA sensitizers are characterized by their higher stability, electron-donating capacity, and aggregation resistance, which render them suitable candidates for DSSC applications14.In the unprecedented dilemma of the ever-growing shortage of energy resources and compelling energy demands, dye-sensitizers are considered the holy grail of renewables and the core of dye-sensitized solar cells (DSSCs) because of their availability and pollution-free nature2 conduction band. Furthermore, TPA compounds can inhibit charge carriers’ recombination of the redox couple  linked to different 2-cyano-acrylamide acceptors and phenyl acetonitrile units. Such D–π–A SFA-5–8 models have been schematically represented in Fig. 2, and amide (CONH) are of great importance in organic synthesis due to their strong accepting ability26. Hence, 2-cyano-acrylamide acceptor and phenyl acetonitrile units were chosen because they possess the good electron-accepting ability and, consequently, facilitate effective interaction with TiO227. In this connection, the development of novel acceptors/anchors for dye-sensitizers is a very crucial task to enhance the performance of DSSCs. Accordingly, higher efficiencies of approximately 5.53–7.56% have been achieved by our synthesized SFA-5–8 sensitizers compared to that reported for the standard N719 dye (7.25%).Generally, TPA-based sensitizers can reduce aggregation and allow the interfacial electron injection of excited dye molecules into the TiOmentclass2pt{minimd6 as a solvent at 400 MHz (1H NMR) and 100 MHz (13C NMR) with an internal standard (TMS), and chemical shifts are given as δ/ppm. UV–Visible spectra were measured by using the high-performance double beam spectrophotometer (T80 series). A Thermo DSQ II spectrometer was used to record mass analyses. The Perkin Elmer 2400 analyzer was used to collect data for the elemental analysis. Finally, in the accompanying information file, all instruments and DSSCs fabrications were thoroughly discussed.The detergents, chemicals, and solvents necessary for the chemical reactions and synthetic procedures were purchased from Sigma-Aldrich, TCI America, and Alfa Aesar, respectively, and utilized exactly as supplied. The measured melting points (uncorrected) are just in degrees Celsius, employing a Gallenkamp electric melting point instrument. A Thermo Scientific Nicolet iS10 FTIR spectrometer was used for identifying the IR spectra (KBr). Nuclear magnetic resonance (NMR) spectra were obtained on a Bruker NMR spectrometer using DMSO-6 has been synthesized through two reactions, firstly phosphonium salt and 2-formylthiophene react via Wittig reaction under alkaline conditions (t-BuOK), At 50 mL two-neck RB flask, 19 mL of freshly distilled POCl3 was added dropwise to the (20 mL) of a stirred solution of dry DMF at 0 °C under argon atmosphere until colored Vilsmeier salt completely precipitates. Then a solution of 5 vinyl)aniline dissolved in 10 mL DMF) was added to the reaction mixture drop by drop with continuous stirring for 1 h. The temperature was increased to 120 °C for 2 h, then the mixture was stirred overnight at room temperature. After completion of the reaction, the mixture was poured into 100 mL of ice-cold water and the pH was adjusted to alkaline by adding saturated sodium acetate solution. The solid product was collected by filtration and synthesized as a previously reported procedure28. The targeted SFA-5–8 sensitizers were synthesized via a Knoevenagel reaction that was applied on methylene compounds [such as 2-(4-nitrophenyl)acetonitrile] and different cyanoacetamide derivatives 7a–c30.Thiophene-2-carbaldehyde compound 7a)  and 5-(4-(diphenylamino)styryl)-thiophene-2-carbaldehyde (6)  were in 50 mL methanol in a round-bottomed flask. To this reaction mixture, drops of 1,8-diazabicyclo[5.4.0]undec-7-ene (DPU) were added, then the contents of the flask were heated for 5 h at 80 °C. The reaction was cooled at room temperature and the mixture was poured into ice water and neutralized with diluted 1 M HCl to yield reddish-brown compound. Yield = 75%, m.p. = 196–198 °C. IR (KBr): νmax 2922 and 2854 (C–H), 2209 (C≡N), 1612 cm−1 (C=C). 1H NMR (DMSO-d6): δ 6.97 , 7.20–7.29 , 7.45 , 7.56–7.64 , 7.94 , 8.15 ppm . 13C NMR (DMSO-d6): δ 111.32, 119.61, 120.75, 121.49, 126.43 (2C), 126.74, 126.81, 127.09, 127.49, 127.78 (2C), 128.73 (4C), 133.35 (4C), 134.51 (2C), 134.81 (2C), 139.90, 146.49 (2C), 147.46, 148.60, 155.13 (2C), 156.31, 160.93 ppm. Analysis calcd. for C33H23N3O2S (525.15): C, 75.41; H, 4.41; N, 7.99%. Found: C, 75.21; H, 4.50; N, 7.87%.2-(4-Nitrophenyl)acetonitrile ((6)  and 2-cyano-N-(4-nitrophenyl)acetamide  were dissolved in methanol (15 mL). Acetic acid (0.20 mL) was added to the mixture, reflux was continued for 18 h and then cooled to room temperature. The solid that obtained was purified using silica gel column chromatography to obtain dark brown solid compound. Yield = 88%, m.p. = 190–192 °C. IR (KBr): νmax 3330 (N–H), 2222 (C≡N), 1675 cm−1 (C = O). 1H NMR (DMSO-d6): δ 6.96 , 7.74 , 7.90 , 8.05–8.14 , 8.21 , 8.24 , 8.46 , 8.49 , 8.93  ppm. 13C NMR (DMSO-d6): δ 112.05, 120.23 (2C), 121.11, 122.35, 126.05, 126.36 (4C), 126.43 (2C), 126.71, 127.10 (4C), 127.39, 130.03, 134.13, 134.42 (3C), 140.03, 147.08 (2C), 148.20, 149.28, 150.29 (2C), 154.74, 155.97, 156.04, 160.54 ppm. Analysis calcd. for C34H24N4O3S (568.16): C, 71.81; H, 4.25; N, 9.85%. Found: C, 71.76; H, 4.18; N, 9.95%.5-(4-(Diphenylamino)styryl)thiophene-2-carbaldehyde 6) and 4-(2-cyanoacetamido)benzoic acid (7c)  and three drops of piperidine were added. The solution was refluxed for 3 h. After cooling to room temperature, the mixture was poured into ice water. The brown precipitate was filtered and washed with water. After drying, the precipitate was purified by recrystallization from a mixture of e ethanol and drops of acetic acid. Yield = 69%, m.p. = 172–174 °C. IR (KBr): νmax 3334 (N–H), 2220 (C≡N), 1722 (C=O), 1661 (C=O) cm−1. 1H NMR (DMSO-d6): δ 6.69 , 7.73–7.77 , 7.93 , 8.07 , 8.14 , 8.18 , 8.27 , 8.43 , 8.52 , 13.67 . 13C NMR (DMSO-d6): δ 112.16 (2C), 117.64, 121.09 (2C), 122.35 (4C), 126.06 (2C), 126.73 (2C), 127.40 (2C), 130.80 (2C), 134.15 (2C), 134.44 (2C), 140.24, 148.20 (2C), 149.29, 150.29 (3C), 150.77 (2C), 154.81 (2C), 155.97, 156.36, 169.00 ppm. Analysis calcd. for C35H25N3O3S (567.16): C, 74.06; H, 4.44; N, 7.40%. Found: C, 74.26; H, 4.52; N, 7.53%.To 50 mL ethanol, 0.76 g (2 mmol) of 5-(4-(diphenylamino)styryl)thiophene-2-carbaldehyde  and 2-cyano-N-(pyridin-4-yl)acetamide (7d)  in 15 mL ethanol, 0.2 mL piperidine has been added. The reaction mixture was refluxed for 10 h and the pure sensitizer SFA-8 was separated by filtration after cooling the solution to room temperature for obtaining dark brown crystals. Yield = 80%, m.p. = 184–186 °C. IR (KBr): νmax 3324 (N–H), 2219 (C≡N), 1679 (C=O) cm−1. 1H NMR (DMSO-d6): δ 6.95 , 7.74 , 7.90 , 8.07 , 8.11–8.14 , 8.20 , 8.24 , 8.40 , 8.46 , 8.49 , 8.92 . 13C NMR (DMSO-d6): δ 110.93 (2C), 119.35, 121.15, 126.05 (2C), 126.66, 127.19 (4C), 127.40 (2C), 128.05 (4C), 128.09, 128.16, 128.35, 128.47, 132.97, 134.00, 134.09 (2C), 143.30, 146.12, 148.23 (2C), 154.76, 155.14, 155.95, 168.04 ppm. Analysis calcd. C33H24N4OS (524.17): C, 75.55; H, 4.61; N, 10.68%. Found: C, 75.77, H, 4.51, N, 10.52%.To a suspension of SFA-5–8) are depicted in Figs. 6), including the reaction of 4-((bromotriphenyl-λ5-phosphanyl)methyl)-N,N-diphenylaniline (3) with 2-formylthiophene (4) under Wittig reaction conditions, obtaining the corresponding thiophene bridge (5). The intermediate 5 was then formulated using the standard Vilsmeier-Hack reaction protocol to yield 5-(4-(diphenylamino) styryl)thiophene-2-carbaldehyde (6) with a good overall yield  styryl)thiophene-2-carbaldehyde (6) with 2-(4-nitrophenyl)acetonitrile (7a) and three 2-cyanoacetamide derivatives 7b, 7c and 7d as shown in Fig. SFA-5–8, as well as their intermediates, were extensively purified using column chromatography in addition to the recrystallization process. The structures of the newly synthesized sensitizers and their intermediates were confirmed by various spectroscopic techniques. According to their spectral analysis and elemental investigation, the molecular structure of SFA-5–8 was determined. The IR spectrum of compound SFA-5 revealed characteristic absorption bands of groups, C–H aliphatic at 2922 and 2854 cm−1, and cyano group (C≡N) at 2209 cm−1. Moreover, the stretching vibration ban of vinyl groups (C=C) appeared at 1612 cm−1. Its corresponding 1H NMR spectrum exhibited a doublet signal at δ 6.97 ppm that could be attributed to the two protons of the vinylic group with (J = 12.00 Hz), singlet for the olefinic proton at δ 7.94 ppm. The aromatic and thiophene protons resonate as multiplet, triplet, and doublet signals at (δ 7.20–8.15 ppm). Additionally, the IR spectrum of compound SFA-6 exhibited stretching vibration bands at 3330 cm−1 and 2222 cm−1 due to the N–H and C≡N moieties, respectively. Moreover, a strong band at 1675 cm−1 has been detected that could be assigned to the C=O group. The visual inspection of the corresponding 1HNMR spectral data revealed a singlet peak at 8.93 ppm corresponding vinylic proton. Further, The IR spectrum of SFA-7 indicated a vibration band of (C≡N) group at 2220 cm−1 as well as a strong vibration band at 1722 cm−1 attributed to C=O (COOH). Meanwhile, the 1H NMR spectrum of SFA-7 showed a singlet signal at 13.67 ppm associated with the carboxylic group proton. On the other hand, the 13C NMR spectra revealed a characteristic signal at δ 169.00 ppm which could be linked to the carbon of the COOH group. Finally, the IR spectrum of SFA-8 reported absorption bands at 2219 cm−1 and 1679 cm−1 that can be properly assigned to the stretching vibration modes of (C≡N) and (C = O) groups, respectively. Additionally, the 13CNMR spectra revealed a characteristic signal at δ 168.04 ppm, ascribable to the carbon of the carbonyl group.Afterward, 2-cyanoacetamide derivatives SFA-5–8 sensitizers have been recorded in tetrahydrofuran (THF) solution. Their corresponding data are summarized in Table The absorption spectra of all synthesize 32. It is important to note that sensitizers (SFA-5–8) demonstrate intense absorption peaks in the visible region, specifically for SFA-5 that can be linked to the ICT from triphenylamine donor to the CN, and NO2 acceptors parts of the nitrophenyl acetonitrile unit. That has been changed to SFA-6–8 which, showed transitions to the CN, CO, COOH, NO2, and pyridine over 2-cyanacetamide derivatives. Notably, it has been observed that the absorption peak maxima (λmax = 400–500 nm) in THF solution for all synthesized sensitizers followed the order of SFA-5 < SFA-8 < SFA-7 < SFA-6. That could be linked to the addition of the π-bridge thiophene group to the SFA-5–8 sensitizers that significantly contributed to energy delocalization and larger polarizability, thus enhancing the visible light absorption32. Moreover, the estimated energy gap (E0–0), which was calculated from the beginning of the UV–visible absorption spectrum33, has been minimized with the presence of the π-bridge linkage, which promoted visible light absorption. Those values followed the order of SFA-6 < SFA-7 < SFA-8 < SFA-5. Importantly, the λmax of SFA-5–8 in the visible region appears at 468, 481, 478, and 474 nm, respectively. As expected, electronic density variations between the donating electron and the withdrawing part could imply a bathochromic shift in the internal charge transfer (ICT) band33, thus reporting molar extinction coefficients (ε) of approximately 1.77 × 104 mol−1 cm−1, 2.32 × 104 mol−1 cm−1, 2.22 × 104 mol−1 cm−1, and 1.95 × 104 mol−1 cm−1 for SFA-5–8, respectively, associated with their lowest energy bands. Those mentioned values are significantly higher than those reported for the Ru N719 dyes, indicating better light-harvesting ability33. For comparison, the UV–Vis absorption spectrum of N719 has been reported in Fig. SFA-6–8 was more red-shifted in contrast to its corresponding mono-anchoring SFA-5 dye. This can be attributed to the extension of conjugation length within the synthesized SFA-6-8 molecules, as well as the delocalization across the entire molecule, which could be caused by the addition of an additional anchoring moiety over 2-cyanoacetamide derivatives34. In practical, SFA-6 comprises 4-nitrocyanoacetamide bearing CN, NO2, and CO substitutions that are mainly responsible for the bathochromic shift with the highest ε value of approximately 2.32 × 104 mol−1 cm−1 amongst all studied dyes, as displayed in Fig. 2) moiety in their anchoring function. Furthermore, it could be deduced that SFA-7 has more bathochromic shift by around 4 nm, compared to its SFA-8 counterpart, which is associated with its higher degree of conjugation.It is generally recognized that organic sensitizers show two distinct principal absorption bands at shorter 250–400 nm) and longer (420–600 nm) wavelengths. Note that the noticeable bands at shorter wavelengths could be assigned to the π–π* transitions. As for the strong absorption peak observed in the visible spectrum region (420–600 nm), it could be mainly related to the intramolecular charge transfer (ICT) from the triphenylamine (donor) to the acceptor moieties00 nm andSFA-5–8 anchored on the TiO2 surface. As could be observed, all SFA-5–8 sensitizers showed broadening and blue shifting in the absorption spectra compared to their corresponding spectra recorded in the solution spectrum (250–550 nm). This hypochromic shift is directly related to H-aggregation and strong interaction between the anchoring groups of sensitizers and the semiconductor photoelectrode, which is highly desirable for achieving efficient light-harvesting and promoting the overall efficiency of sensitizers35. In particular, it should be emphasized that the observed spectral broadening for SFA-7 could be extensively beneficial for enhancing the visible light-harvesting ability and enhancing the short circuit current (Jsc). The highest absorbance values exhibited by the SFA-7 sensitizer compared to other sensitizers can be mainly ascribed to the deprotonating of carboxylic acid or the formation of H-aggregates on the TiO2 surface, which lead to lowering the π* energy level of its 4-carboxylcyanacetamide group and thereby broadening its absorption spectrum. That in turn could confirm the good ability of the 4-carboxylcyanoacetamide of the SFA-7 sensitizer to harvest more photons compared to that of the N719 counterpart. Contrarily, SFA-5 showed the weakest absorbance characteristics compared to other 2-cyanoacetamide derivatives of SFA-6–8 sensitizers, thus confirming the direct effect of anchoring and acceptor groups on the absorbance enhancement. Thereby, we believe that including extra anchoring groups in the design of sensitizers could in turn promote the dye adsorption on the TiO2 surface, narrow the spectrum, and most importantly facilitate the electron flow from the excited dyes to the TiO2 conduction band36.Figure SAF-5–8 dyes were established to unravel how the characteristic π-spacer and different anchoring/acceptors moiety thru phenylacetonitrile and cyanoacetamide derivative sensitizers influence the geometry of the targeted dyes and their DSSC photovoltaic efficiency. The details of the calculations are based on Gaussian 09 software37 via B3LYP/6-311 g  sets38. As demonstrated in Fig. SFA-5–8 have been reported. Moreover, their corresponding dihedral angles and bond lengths have been summarized in Table SFA-5–8 dyes show a propeller starburst arrangement and (D–π) dihedral angles of about  with π-bridges that could be of great importance for achieving efficient charge transfer.Computational studies for SFA-5–8 sensitizers. Meanwhile, DFT calculations showed that SFA-5–8 structures possessed higher dihedral angles that are close to 180° or equal to zero. Such values could reflect better coplanarity configurations and favorable conjugation between the thiophene-π-spacer and the acceptor moiety38. Indeed, those findings could infer the highest λmax values of the SAF-5–8 structures which is a good indicator of the fast electron transfer to the semiconductor surface, resulting in boosting the overall performance. For further insights, Table SFA-5 as D–π (1.50 Å), π–π (1.34 Å), π–A (1.50 Å), SFA-6 as D–π (1.50 Å), π–π (1.34 Å), π–A (1.34 Å), SFA-7 as D–π (1.42 Å), π–π (1.34 Å), π–A (1.32 Å) and SFA-8 as D–π (1.50 Å), π–π (1.36 Å), π–A (1.44 Å). Because of the steric hindrance between the donor (D), π-bridge, and acceptors units, we can theoretically determine the co-planarity of the compounds through the dihedral angle calculation. Therefore, designated dye sensitizers have a significant potential to avoid unfavorable dye aggregation (π–π). It is well documented that the dye aggregation mainly contributes to the self-quenching of excitation and hence lowers the efficiency of the electron injection. Therefore, even the dye molecules’ structural geometry gave a valuable foresight about the perceptions of their improved photovoltaic performances as reference38. All of these computed findings revealed a strong substantial conjugation impact that could strongly stimulate the electron transfer from the donating TPA moieties to the different electron acceptors over SFA-5–8 structures.Note that inserting the thiophene moiety is advantageous for increasing the conjugation degree of the SFA-5–8 sensitizers, NBO analysis could be of crucial importance. In this context, NBO analysis could study the stabilizing interactions between the filled (donor) and empty (acceptor) orbitals as well as the destabilizing interactions between the filled orbitals39. By using the second-order perturbation approach, the hyper conjugative interaction energy was estimated from a donor (i), an acceptor (j), and the stabilization energy E(2) associated with the delocalization j, i could be estimated as the following equation:qi represents the donor orbital occupancy, F is the off-diagonal. In NBO studies, strong intermolecular hyper conjugative interactions were analyzed by the second-order perturbation theory of the Fock matrix. For SFA-5–8 sensitizers, the higher energy values of E(2) hyper conjugative interactions, the more intensive interaction between an electron donor (TPA moiety) and electron acceptors , means the greater ability to donate tendency from electron donor parts to electron acceptors. The strong stabilization energy for SFA-5 sensitizer is from π* (C16–C17) → π* (C20–C21), π*((C8–C15) → π*(C16–C17), π (C31–C32) → π* (N37–O39) have the highest E(2) values 231.78, 111.85, 68.82 kcal/mol, respectively. This electron transition represents electron transfer across the sensitizer from triphenylamine (donor) to thiophene (system) ring to phenyl acetonitrile (acceptor), indicating effective charge transfer40. Similarly, in the case of SFA-6, important interaction π*(C24–C25 → π*(C22–C23), π*(C35–C36) → π*(C31–C37), and π*(C29–O32) → π*(C27–C28) have the highest E(2) values of 85.04, 36.74 and 30.43 kcal/mol, respectively. For SFA-7, there occurs a strong intramolecular hyper conjugative interaction from D–π–A, represented in π*(C10–C11) → π*(C12–C13), π*(C10–C11) → π*(C9–C14), π*(C40–O41) → π*(C35–C36) and π*(C22–C32) → π*(C20–C21) have the highest E(2) values of 68.43, 50.03, 29.89 and 26.21 respectively. SFA-8 transition π*(C24–C25 → π*(C22–C23) which leads to strong delocalization, π*(C35–C36) → π*(C31–C37) and π*(C29–O32) → π*(C27–C28) showed the highest E(2) values 90.04, 60.78 and 50.98 kcal/mol, respectively.To further elucidate the origin of the intramolecular interactions and give deep insights into the intramolecular electron transfer processes within the synthesized Quantum chemical parameter SFA-5-8.SFA-5–8 sensitizers could be analyzed by utilizing the quantum analysis by calculating several significant factors; including the bandgap energy (E0-0), ionization energy (IP), electron affinity (EA), hardness (η), and softness (s), respectively. With respect Koopmans’ hypothesis framework41, the corresponding data are displayed in Table The chemical reactivity of the SFA-5–8 decreased in a sequence of SFA-7 < SFA-8 < SFA-6 < SFA-5. The smallest E0–0 of the SFA-7 structure could reveal its highest stability and confirm its reactive configuration amongst all studied sensitizers, thus allowing for more dominant excitation between the HOMO and LUMO of the SFA-7 dye molecule. Accordingly, 4-carboxycyanoacetamide SFA-7 sensitizer should be chemically more reactive than other SFA-5, SFA-6, and SFA-8 dyes. On the other hand, hardness (η) and softness (s) are very useful parameters that could reflect the system's reactivity. On one hand, hardness could assess the maximal electromotive force between donors and electron acceptors within the same molecule. In this regard, accurate determination of energy gaps can be used to classify a substance as either hard or soft. For instance, a high HOMO–LUMO gap means a hard species, whereas a low HOMO–LUMO gap indicates a soft moiety41. Importantly, the global hardness of a pure substance was linked to its stability and reactivity. The reactivity is inversely linked to global hardness, while the stability is closely linked to it41. Further insights, the global hardness for SFA-5–8 sensitizers was reported to be η = 1.13 > 1.10 > 1.05 > 1.09 eV, respectively. The lower hardness (η) value could be appropriate for allowing an effective charge transfer within the dye molecules. Thereby, SFA-7 could show efficient charge transfer features when compared to its corresponding sensitizes (SFA-5–8). Further, the key photovoltaic parameters such as ∆Ginj., ∆Greg., and ∆Grec. were estimated as shown in Eqs.  of theTiO2 molecule. That in turn could enhance the Jsc values and promote the DSSC efficiency. It could be observed that all driving force values (ΔGinj) of the synthesized SFA-5–8 dyes are negative, which is ideal for hole injection. Most importantly, SFA-7 had a greater value than the other TPA dyes, confirming that adding one additional acceptor and anchoring moieties  to the dye molecule could facilitate the charge transfer features, thus better DSSC performance42. For comparison, the predictable dye regeneration values are listed in Table SFA-5 (0.76 eV) > SFA-6 (0.50 eV) > SFA-8 (0.45 eV) > SFA-7 (0.34 eV). As reported in Table SFA-7 has the smallest ΔGreg. value of ~ 0.10 eV, demonstrating the maximum dye regeneration capability. Those who reported ΔGrec. values have been decreased as the following SFA-7 (1.34 eV) < SFA-8 (1.45 eV) < SFA-6 (1.50 eV) < SFA-5 (1.76 eV). When SFA6–8 sensitizers were injected, three fractions carrying the 2-cyanoacrylamide derivatives with different acceptors had been proposed which could defeat the charge recombination to a specific limit. In this context, SFA-7 has reported the lowest charge recombination value (ΔGrec) of about 1.34 eV in comparison with other structures in the same class. Accordingly, based on the calculated outcomes of the aforementioned factors ΔGinj, ΔGreg, and ΔGrec, SFA-7 could confirm its superiority as a high efficient dye for DSSCs applications. Moreover, those previous findings could suggest that phenyl acetonitrile and 2-cyanonacetamide of SFA-5–8 compounds might be also viable dyes as good choices for DSSC.ΔGSFA-5–8 sensitizers to the TiO2 molecule, as well as to investigate the electron regeneration of the dyes via calculating the ground and excited oxidation potentials of the newly synthesized sensitizers42(SFA-5–8). These values represent the key criterion to evaluate the suitability of an organic dye for DSSC applications and provide a deeper insight into the thermodynamic driving force of electron injection and dye regeneration. Such corresponding values are depicted in Fig. 42. GSOP values for the studied SFA-5–8 dyes were found to be: SFA-5 (− 5.94 eV), SFA-6 (− 5.78 eV), SFA-7 (− 5.58 eV), and SFA-8 (− 5.68 eV). The HOMO of sensitizers is more negative than the value of 43.Cyclic voltammetry (CV) experiments were conducted to determine the electron transfer feasibility from the (SFA-5–8) was estimated from the above-mentioned equation using both the GSOP and E0-0 . The ESOP energy levels are lying between − 3.48 and − 3.65 eV for the sensitizers: SFA-5 (− 3.65 eV), SFA-6 (− 3.55 eV), SFA-7 (− 3.41 eV), and SFA-8 (− 3.48 eV), respectively. Those values are higher than that of the TiO2 conduction band. It is worth mentioning that the LUMO level of the di-anchoring dyes completely changed for the mono-anchoring counterparts, associated with the conjugation length and the electron-withdrawing character of the synthesized dyes acceptors as well as the HOMO–LUMO minimized band gaps44. As expected SFA-6–8 molecules with nitrocyanoacetamide, carboxycyanoacetamide, and pyridinyl cyanoacetamide as strong acceptor moieties showed the smallest bandgap values compared to that of SFA-5 with the phenylacetonitrile segment. That could be a direct evidence on the highest degree of conjugation between the electron-accepting  units and donor parts. As could be noticed, all ESOP data are energetically less positive than that of the TiO2 CB potential (− 4.2 eV), unraveling the efficient injection of the electron to the TiO2 edge. Amongst all synthesized dyes containing cyanoacetamide segments, SFA-7 contains the strongest electron-withdrawing group (COOH) than pyridyl cyanoacetamide of SFA-8 and 4-nitrocyanoacetamide in SFA-6. That in turn could imply a deficient driving force for the electron transfer by possessing the smallest E0-0 values and ensuring efficient light capture. All the sensitizers (SFA-5–8) could meet the thermodynamics prerequisite, hence rendering them suitable to be used as effective sensitizers for TiO2-based DSSCs.Additionally, ESOP of sensitizers SFA-5–8 sensitizers using the B3LYP hybrid method in parallel with the d-polarized 6–311G basis implemented in the Gaussian09 program37. Figure 45. Intramolecular charge transfer (ICT) from the dye HOMO level (depending on donor part) to its LUMO level (depending on acceptor part) is considered one of the most important features for achieving effective charge separation and better electron injection. For SFA-5–8 sensitizers, there is an effective charge separation between the HOMOs and the LUMOs levels. For the SFA-5 sensitizer, the electron density of HOMO was primarily localized on the donor parts (triphenylamine and thiophene ring), whereas the electron density of LUMO was largely localized on 4-nitroacetonitrile acceptor moiety (CN and NO2). However, this case was different for the SFA-6 as the 4-nitrocyanoacetamide insertion had enlarged the conjugative system without impairing the coplanarity of the entire molecule. As such, the HOMO electron density was localized on triphenylamine donor part, but the LUMO counterpart shows a clear shift in the electron density towards the acceptor moieties . Noteworthy, the introduction 4-carboxylcyanoacetamide into the SFA-7 dye could facilitate the electron transfer from the TPA donor side and thiophene moieties to the acceptors part that localized on CN, CO, and COOH segments. As for the SFA-8, the electronic distribution of its HOMO is mainly distributed over the donor and π-spacer, however, its LUMO orbitals are distributed mainly on the electron acceptor units of the 4-pyridylcyanoacetamide, resulting in strong electronic interaction with TiO2 surface. It is not surprising to mention that the intramolecular charge transfer (ICT) from various donors to phenyl acrylonitrile/2-cyanoacetamide donors across all sensitizer moieties via the π-spacer had facilitated the HOMOs-LUMOs spatial level separation upon light irradiation. Accordingly, the electron injection from the excited sensitizer dyes to the TiO2 surface had significantly enhanced.DFT calculations were performed on 46 of triphenylamine of SFA-5–8 dyes, which can be obtained from the cube file of the Gaussian job37. The effect of donor–acceptor groups was analyzed by inspecting the different HOMO–LUMO levels and (MEPS) of all SFA-5–8 sensitizers , and (NO2) groups. On the other hand, the negative charge of SFA-6 molecules with cyanoacetamide moieties were localized over cyano, carbonyl, and nitro groups. As for the SFA-7 dye, its negative charge was localized on the cyano group, and carbonyl attached to the COOH group. Furthermore, the negative charge of SFA-8 is founded to be linked to CN, CO, as well as the nitrogen of the pyridine ring. From further insights, the blue positive region of the MEP map was localized over the donor moieties such as the triphenylamine ring, π- conjugation system, and thiophene ring regions, thus demonstrating their favorite sites for nucleophilic attack. Indeed, analyzing all previous MEP features of all synthesized SFA-5–8 sensitizers revealed the electron feasibility for possible interaction with another group of atoms. These findings can reasonably give an indication of the ICT nature of all synthesized SFA-5–8 sensitizers when they are adsorbed on the TiO2 surface.Molecular electrostatic potentials (MEP) is one of the efficient approaches to identify the internal charge transfer (ICT) property of the entire organic molecules between HOMO–LUMO levelsers Fig. . On one 2 fabrication process was thoroughly provided in the “TiO(SFA-5–8) side by side with the charge carrier efficiency of the adsorbed dyes on the TiO2 surface, the incident photon-to-electron conversion efficiency (IPCE) was extensively measured under no external bias, as depicted in Fig. 47. As shown in Fig. IPCE values of all DSSC based on SFA-5–8 sensitizers showed maximum IPCE peaks reaching (58–68%) at a range of 300–800 nm, confirming the vital role of pheylacetonitrile and 2-cyanoacetamide derivatives present in all synthesized dyes on enhancing the photoelectrochemical performance. Specifically, SFA-7 reported the highest IPCE peak of above 68% at ~ 300–800 nm along with the enhanced JSC value amongst all studied dyes. This can be explained based on the fact that incorporating a strong electron-withdrawing carboxylic group (COOH) of the 4-carboxylcyanoacetamide segment to other anchoring CO and CN moieties of SFA-7 contributed largely to maintaining the highest IPCE value. That was even higher than those reported for N719 dye, thus revealing higher photocatalytic efficiency of 4%. As for the SFA-8 sensitizer, carboxylic group replacement with a pyridyl ring has minimized its electron light-harvesting ability and hindered its electron injection, thus reporting a lower efficiency in contrast to the SFA-7 sensitizer. Although 4-nitrocyanoacetamide SFA-6 has the same skeleton of carboxyl and pyridyl 2-cyanoacetamide sensitizers as SFA-7–8 analog, the IPCE peak of the SFA-6 was found to be lower than its corresponding SFA-7 and SFA-8 sensitizers, mainly highlighting the strength of the electron-withdrawing groups48. This lower efficiency could be attributed to the presence of nitro group (NO2) that strongly promoted the dye aggregation and hindered the IPCE values, as confirmed in Fig. SFA-5 offered weaker absorption with a less covered surface on the TiO2 surface compared to other SFA-6–8 sensitizers, thus influencing the overall yield and retard the electron injection processes49. The JscIPCE values integrated from the IPCE spectra are in good consistency with the Jsc values measured in the J–V measurements. Consequently, the cell with dye SFA-7 gives the highest and broadest IPCE spectrum, confirming its highest Jsc. The improved IPCE responses are consistent with the improved Jsc49. Furthermore, the IPCE spectra of all SFA-5–8 sensitizers and N719 are in good agreement with their UV–vis absorption spectra on the TiO2 surface.To further evaluate the Photovoltaic enhancement and assess the electron injection efficiency of all sensitized devices JSC), open-circuit photovoltage (VOC), fill factor (FF), and power conversion efficiency (ηcell). The photocurrent density–voltage spectra for N719, phenylacetonitrile (SFA-5), and 2-cyanoacetamide sensitizers (SFA-6–8) are depicted in Fig. IPCE response of 4-carboxylcyanoacetamide SFA-7 sensitizer was translated into the highest Jsc of approximately (17.51 mA/cm2) in comparison with the reported Jsc values of N719 (17.00 mA/cm2), SFA-8 (16.78 mA/cm2), SFA-6 (16.10 mA/cm2), and SFA-5 (15.16 mA/cm2) counterparts. Such short circuit current enhancement of SFA-7 molecule could be mainly linked to better electron injection efficiency to TiO2 conduction band compared to other sensitizers. That was also confirmed by the highest value of its LUMO levels. In a bid to further examine the photovoltaic performance of the synthesized cells, open-circuit voltage and fill factor could give insights into the photoelectrochemical performance of the synthesized sensitizers. For instance, N719 and SFA-5–8 dyes yielded power conversion efficiency (% ηcell) of 7.25 (Voc = 0.70 V and FF = 0.61), 5.53 (Voc = 0.63 V and FF = 0.69), 5.77 (Voc = 0.64 V and FF = 0.56), 7.56 (Voc = 0.72 V and FF = 0.60), and 6.43 (Voc = 0.65 V and FF = 0.59), respectively. The VOC of sensitizers follows the order of SFA-7 > N719 > SFA-8 > SFA-6 > SFA-5.Table SFA-5, the anchoring occurs through the cyano group (CN) working as a withdrawing group, and the coordination between the nitro group and the TiO2 surface causes optical bleaching of the light absorption, increases the dye/TiO2 coupling, which provides the process of anchoring on the surface TiO2. Nitrophenyl acetonitrile sensitizers (SFA-5) showed the lowest efficiency attributed to the weakest acceptor moieties compared to other 2-cyanoacetamides sensitizers (SFA-6–8) and showed the lowest values of JSC and VOC values. For SFA-6, by replacing the phenyl acetonitrile by 4-nitrocyanoacetamide, the anchoring process occurs through a carbonyl group (CO), (NH), (NO2), and (CN) groups that work as excellent withdrawing groups which provide the process of anchoring on the surface TiO2, increasing the number of anchoring function groups enhanced the efficiency than anchoring by only (CN) and (NO2) in SFA-5, the highest JSC, and better VOC values were reported for the 4-carboxylcyanoacetamide SFA-7 sensitizer associated with a higher photoconversion efficiency of about 7.56%, thus outperforming the best-reported dyes which mainly related to the various strong acceptor's group, carboxyl group plays a great role in surface adsorption on TiO2 addition to act as electron acceptors, given excellent electron-withdrawing capabilities relative to the Brønsted acid sites employed for the adsorption of carboxylates. The presence of a second strong anchoring group (COOH) as an extra acceptor addition to the various anchors across the 2-cyanoacetamide segment (CO), (CN), and (NH) act as bifunctionality of acceptor and electron-withdrawing groups which seems to enhance the performance of the sensitized cells and the electron injection into the conduction band of TiO2 through H-aggregates50. This is not surprising as the photoconversion efficiency of the SFA-7 sensitizer was raised by 4% with respect to N719. Finally, for SFA-8, using 4-pyridylcyanoacetamide for the first time as a new bifunctionality anchoring and electron withdrawing system, by the formation of coordination bonds between the nitrogen atom in the pyridyl ring of the Lewis acidic sites of the TiO2 surface leads to efficient electron injection, addition to the electron-withdrawing groups across the 2-cyanocetamide segment . Moreover, SFA-6 and SFA-8 sensitizers carrying 2-cyanoacetamide segments showed lower efficiency than SFA-7, which mainly related to the strength of the anchoring group in the order of COOH > pyridyl > NO2. Thus, Jsc and VOC values have been changed accordingly. Based on the above findings, it could be noticed that the experimental results are in accordance with the theoretical predictions.As stated in Table SFA-5–8 sensitizers with the enhanced photocatalytic performance, electrochemical impedance spectroscopy (EIS) was investigated to effectively determine the charge transfer resistance (RCT) and the interfacial capacitance at the TiO2 electrode/dye/electrolyte and Pt/electrolyte interfaces51. Figure SFA-5–8 and N719 sensitizers. As depicted in Fig. FF. On the other hand, a large semicircle was observed in the middle frequency regime that could be originated from charge transfer resistance (RCT) from TiO2 molecule to the electrolyte solution, which is directly related to the Voc. According to Nyquist plots, the semicircle radius of all sensitized SFA-5–8 cells follow the order of SFA-7 > N719 > SFA-8 > SFA-6 > SFA-5. It is worth noting that the Rrec of 4-carboxylcyanoacetamie SFA-7 is larger than that of the N719 counterpart, indicating that the charge carriers recombination process was retarded upon sensitization. In addition, the high Rrec value of the SFA-7 molecule upon sensitization could be mainly ascribed to the SFA-7 structure containing various acceptors and anchoring moieties that are favorable for retarding charge recombination process. To this end, Nyquist plots unravel that sensitization by 2-cyanoacetamide sensitizers could be beneficial for reducing dark current and suppressing charge recombination at the TiO2/dye/electrolyte interface52.To better correlate the charge dynamic characteristics of all synthesized eff = 1/2πf)52, where t represents the electron lifetime injected into TiO2 and f is the mid-frequency peak in bode plots, which is directly related to the electron lifetimes. Notably, the electron lifetime that is mainly related to Voc was determined for all synthesized SFA-5–8 molecules using Bode frequency plots. The values of the mid-frequency peaks of the bode plots followed the order of SFA-7 > N719 > SFA-8 > SFA-6 > SFA-5, corresponding to electron lifetimes in the sequence of SFA-5 < SFA-6 < SFA-8 < SFA-7. Similarly, the corresponding electron lifetimes were found to be 4.48, 3.25, 2.58, 2.14, and 1.26 ms, respectively, in agreement with Voc values. For the sensitized SFA-5–8 and N719 dyes, the electron lifetimes follow the order of SFA-7 > N719 > SFA-8 > SFA-6 > SFA-5, respectively, which lead to a significant enhancement in photocurrent and photovoltage and considerably higher cell efficiency53.On the other hand, EIS could be represented using Bode frequency plots, as illustrated in Fig. SFA-5–8 with different auxiliary donors and acceptors. We studied how the presence of a thiophene spacer and different auxiliary acceptors  affected the photophysical and electrochemical performance of DSSC devices. DFT and TD-DFT were used to explore the photophysical and electronic structures of all studied SFA-5–8 organic dyes. The molecular orbital energy levels of SFA-5 sensitizers possessed an appropriate thermodynamic driving force that was sufficiently suitable for electron injection from the LUMOs into the TiO2 conduction band. The calculated studies of the dihedral angle and NBO of SFA-5–8 revealed an efficient ICT across sensitizers from the donor to acceptor parts. The higher electron acceptance and lower chemical hardness values of SFA-5–8 molecules suggested excellent photoelectric conversion performance. Further, we measured the photovoltaic properties to properly investigate the direct effect of various anchoring units on the overall performance of the DSSCs. The overall photoconversion efficiency of DSSCs for all SFA-5–8 sensitizers was approximately 5.53–7.56%. The number of sensitizers loaded into the TiO2 surface is in accord with the PCE values. Remarkably, SFA-7 showed superior photoconversion efficiency associated with the highest Jsc and Voc which could be attributed to reduced recombination rates of charge carriers and the increased electron lifetime. The sensitizations of the new SFA-5–8 sensitizers were investigated in comparison to the N719 dye. Interestingly, the optimized SFA-7 attained a higher PCE by approximately 4% over that of N719 sensitized one. This splendid activity can be ascribed to the excess anchoring groups of the synthesized dyes that could lead to a longer blocking layer that minimizes charge recombination. Such breakup of π-stacked aggregates could improve the electron injection yield, rendering SFA-5–8 sensitizers superior candidates for applied DSSC applications.In summary, we have synthesized four innovative phenylacetonitrile and 2-cyanoacetamide derivatives of dyes Supplementary Information.


Candidatus Phytoplasma pyri’, causing the pear decline disease. Specific repellents are a potential method for controlling this pest and reducing the spreading of phytoplasma pear in orchards. Little is known about the chemical ecology of these insects. Based on recent literature, we tested the behavioral reactions of Cacopsylla pyri and Cacopsylla pyricola to the major synthetic compounds of clove essential oil in olfactometer experiments. In addition, we developed new nanofiber-based repellents dispensers and evaluated their repellent and host-odor masking activity in the laboratory and later in the field. Results demonstrate that only the synthetic mixture of the three major compounds of the clove essential was repellent to both psyllids species. When volatiles were formulated in nanofibers, they were not only repellent but could mask the odors of pear plants, disrupting the insects from finding the host. In the field, no differences in psyllids captures were observed in color-attractive sticky traps with repellent-loaded nanofibers or nanofibers without repellent. Our study also evaluated the release rates of volatiles from the nanoformulation. We discussed using nanofibers as volatile dispensers and the improvements necessary to use repellents as a management tool for pear psyllids in the field.Pear psyllids are responsible for transmitting the pathogen ‘Candidatus Phytoplasma pyri’ causing pear decline. Based on earlier reports, we tested the behavioral activity of the major synthetic compounds of clove essential oil  against Cacopsylla pyri and C. pyricola. Of six mixtures tested in olfactometer assays, a formulation consisting of three specific compounds (M6 mixture) demonstrated a repellent effect on both psyllid species. In addition, this formulation masked the odor of the host Pyrus communis cv. Williams Christ, disturbing the host finding ability of C. pyri. Electrospun fibers were produced with biocompatible polymers poly(ε-caprolactone), cellulose acetate, and solvents formic acid and acetic acid, loaded with the repellent mixture to test their efficacy as dispensers of repellents in laboratory and field. The fibers produced were repellent to C. pyri and effectively masked the odors of pear plants in olfactometer tests. In a pear orchard, we compared the captures of pear psyllids in green-colored attractive traps treated with nanofibers loaded with M6 mixture or unloaded nanofibers (blank). The result showed no differences in the captures of C. pyri between treatments. The release rates of volatiles from the fibers were evaluated weekly over 56 days. The fibers were able to entrap the major compound of the M6 mixture, eugenol, but the release rates were significantly reduced after 21 days. Our results suggest that biodegradable dispensers could be produced with electrospinning, but further improvements are necessary to use repellents as a management tool for pear psyllids in the field.Pear psyllids are the main vectors of the pathogen ‘ Candidatus Phytoplasma pyri’, a phytoplasma that belongs to the Apple proliferation group (16SrX) ; (16SrX) . The traC. pyri) . In Nortn vector ,5. Transin Italy , suggestC. pyri, influencing movement and host choice [C. pyri population [Most phytophagous insects are specialists , who uset choice . Pest mapulation . Behaviopulation ,12. Pastpulation ; howeverpulation ,15, therpulation , a numbeBiopesticides encompass a large number of technologies, from microbial organisms to botanicals. Among the botanicals, essential oils are a significant category that began to develop with research in the 1980s ; they arC. chinensis and observed that, in the field, clove essential oil treatment could reduce the number of nymphs in a concentration-dependent manner; however, there is no report on the possible repellent activity against pear psyllids (C. pyri and C. pyricola). Since clove is a less related plant to pear and has less similar odors, we predicted that they are more likely to repel C. pyri and C. pyricola. The main component of clove essential oil is notably eugenol; however, there are differences in the composition of its constituents; those differences may be caused by the variation of vegetative state, growing season, and the places of origin [Clove essential oil has been widely studied for its insecticidal and repellent activities against many species of pests ,25,26, sf origin ,36,37, mf origin .Nanotextiles are emerging as the most potent tool for future sustainable agricultural production advances. The innovative application of nanotechnology, specifically polymeric nanofibers as vehicles for dispensing semiochemicals, was reviewed by Czarnobai De Jorge and Gross (2021) . These fC. pyri and C. pyricola and to test if the formulation and incorporation of synthetic volatiles of clove in nanofibers can be efficiently applied repellents for pear psyllids in the field. The release rates of the volatiles from nanofibers were also determined.Therefore, this study aims to evaluate the effect of clove essential oil major constituents against C. pyri used for olfactometer trails were sampled daily from Pyrus communis cv. Williams-Christ trees at the Julius Kühn-Institut (JKI) experimental orchard in Dossenheim, Germany. Insects were captured using the beating tray method, according to Weintraub and Gross (2013) [C. pyricola individuals were obtained from a laboratory culture maintained at the Julius Kühn-Institut (JKI) ; they were maintained without exposure to insecticides on healthy potted Pyrus communis cv. Williams Christ plants in a 47.5 × 47.5 × 93 cm rearing cages  in a climatic chamber with 20 °C day and 15 °C night temperatures under long-day conditions (L16:D8) and 55% relative humidity [Adults of s (2013) , and thes (2013) ,41. C. phumidity .All volatiles examined in this study were selected from the literature . The com® ≥ 99% purity, LC-MS Grade, Carl Roth), formic acid . PCL/CA (1:1) were dissolved in acetic acid: formic acid mix (1:1). The concentration was kept at 15% (w/v) with respect to solvent. The solutions were made by continuously stirring the mixture overnight at 1500 rpm with a magnetic stirrer at room temperature to obtain a completely homogenous solution. The concentration of the synthetic mixture in the solutions was 5% and 10% (v/v).Poly-(ε-caprolacton) , cellulose acetate , acetic acid  and an applied voltage of 12 kV. The resulting fibers were kept under a ventilated hood overnight for complete solvent evaporation and stored at −20 °C until performing the tests.C. pruni to host odors. The Y-olfactometer was placed in a dark room and was illuminated with a LED magnifying light of 280 lx  45 cm above the middle of the olfactometer. All tests were performed between 12:00 a.m; moreover 6:00 p.m. at room temperature and humidity (20–26 °C and 30–35% RH). On cloudy and rainy days, no tests were performed, since psyllid motivation is lower [C. pyri females and C. pyricola females from lab culture were tested. Single psyllids were collected in 1.5 mL plastic vials and left to starve overnight in the refrigerator kept at 6 °C. About an hour before the experiment, insects were transferred to room temperature. A single female was introduced at the base of the Y-tube olfactometer and observed for 5 min. Only the psyllids were counted that entered one of the test arms and passed 2/3 of the tube, remaining there for at least 30 s. Individuals that did not make a choice within 5 min were recorded as “not responding.” After each experiment, all olfactometer parts were rinsed with ethanol (70%) and heated at 230 °C for three hours with the exception of the plastic valves (60 °C for 2 h). After five repetitions, the side on which the treatment was presented was swapped to avoid positional bias. A minimum of 26 individuals were tested in each trial. The following experiments were conducted:(a)C. pyri and C. pyricola. A stock solution of all mixtures To observe the host odor masking effect of the M6 mixture of clove volatiles in the presence of pear tree volatiles, one potted pear plant cv. ‘Williams-Christ’ (50 cm) was carefully wrapped in oven plastic bags  and connected to one of the test arms. In the other arm an empty oven bag was used as control. Air was pumped to the wrapped plants and empty oven bag for 30 min before the experiment to stabilize the system. The airflow passing at the outlet of the oven bags and entering each olfactometer arm was controlled by a flowmeter  and adjusted with plastic valves. In the olfactometer arm contained the pear volatiles, a filter paper containing the M6 mixture, as described above, was added, and the other arm was supplied only with solvent.(c)For the test with volatiles encapsulated into nanofibers, a 3 mg piece of nanofibers loaded with 5% of the volatile M6 mix was offered in one olfactometer arm. As a control, purified air was pumped into the other arm. The masking effect of the nanoformulation was also tested as above: in one olfactometer arm, nanofiber + M6 was offered with the pear volatiles, and the other arm was supplied only with air.To determine female pear psyllid’s response to synthetic volatiles contained in clove essential oil, olfactometer tests were performed. A dynamic glass Y-shaped olfactometer  was mounted on a board at an angle of 40° from the horizontal plane. The experiments were conducted as previously reported , to inveis lower . The airis lower ,44. The Field experiments were conducted to observe if attractive stick traps equipped with nanofiber formulated with the repellent mixture (M6) would reduce the catches of psyllids in comparison with traps equipped with blank nanofiber. The test was performed in a non-commercial pear orchard in Germany . The traps were fabricated from transparent 0.02-mm-thick rigid vinyl plastic cylinders, 9 cm in diameter × 25 cm in length, coated with a thin layer of insect glue, provided by Insect Services GmbH, Berlin. Traps were designed with 16 holes (1.5 cm diameter) evenly distributed (5 cm distance between each other) a. Test tTraps were evaluated weekly for six weeks between 21 June 2021 and 2 August 2021. Captured psyllids were identified, counted, and removed from the traps.A six-chamber unit with the capability to analyze six samples at once was employed in the experiment , Markes International GmbH, Offenbach, Germany). Nanofiber samples containing 10% M6 were cut in small pieces, each piece 30 ± 0.1 mg and then placed into the individual chambers made from stainless steel (n = 6); they were incubated at an optimal temperature of 24 °C and purged with 100 ± 0.1 mL/min constant flow with high-purity synthetic air . Following an equilibration period, clean sorbent tubes  were connected to the outlet of each individual micro-chamber to collect the volatiles purged from the nanofibers sample. Volatiles were collected for one minute. After sampling, the sorbent tubes were sealed tightly by Swagelok caps for storage. Immediately before the tests n-dodecane (50 ng) was added in the sorbent tubes as an internal standard. Subsequently, tubes were loaded onto an automated thermal desorption prior to analysis by GC-MS (details see below). To observe the release rates of the M6 VOCs from nanofibers, new fibers were left under a laboratory hood after the sampling start and sampled every seven days for 56 days, as described above.m/z.An automated thermal desorption system  connected to a gas chromatograph coupled with a mass spectrometer (GC–MS)  was usedEvaluation of GC–MS chromatograms was carried out using “Automated Mass spectral Deconvolution and Identification System”  following the protocol in Gross et al. (2019) . The comp values adjustment by Tukey.The data evaluation was conducted with the program R (Version 1.2.5033). All figures were generated using the “ggplot2” package. Binomial tests were used to determine the significance of choice between treatments odor vs. control in two-choice Y-olfactometer assays. Non-responder were excluded from this analysis. For field trials, the averages of psyllids collected by trap were compared using generalized linear models (GLMs), assuming a quasi-Poisson distribution (count data with overdispersion). We considered “week” and “trap type” as a fixed factors in the model. Using a deviation analysis , we investigated whether the factor ‘trap’ significantly influenced the number of insects oriented towards the traps. Interactions between the temporal repetitions of experiments and trap type “week: trap” were included in the model as fixed factors. The evaluations of the release rate of volatiles from nanofibers were performed with GLM quasi-Poisson model, considering “days” of exposure of nanofibers as a fixed factor. The data was log-transformed for the analysis. The deviation analysis was performed to evaluate the impact of exposure time “days” on the release rates . Fitted models were tested for the field experiment, and nanofibers release rates with a residual diagnostic for hierarchical (multi-level/mixed) regression models. Multiple pairwise comparisons were calculated with estimated marginal means and 95% confidence intervals with the function emmeans from the “emmeans” package  and p vaC. pyricola were significantly repelled by M5 and M6 ; however, the other blends and single compounds vs. methanol did not affect their behavior . In this case, β-caryophyllene and eugenyl acetate alone were not repellent to C. pyri females (C. pyri than in C. pyricola (80% and 68%). We observed that M6 was effective in repelling both psyllid species in choice assays .1,53 = 0.83; p = 0.34). Statistically, C. pyri catches on traps equipped with nanofibers and M6, and the blank ones were similar; moreover, the interaction between trap type capture (blank and M6) and week  was not significant. According to the model, the only significant factor was week . The number of C. pyri captured in traps was significantly influenced over time . There was no difference between the amount of released eugenol from new nanofibers and after seven and 14 days of exposure , but already reduced in relation to the rates of new nanofibers (p = 0.027). The release rates of eugenol decreased after 28 d and remained constant until the end of the tests (No traces of the solvents acetic acid and formic acid used to produce the fibers were detected in the chromatograms. The compound β-caryophyllene was not detected in the fibers, and eugenyl acetate was only detected during the first two weeks of tests. Since eugenol was consistently detected in all the samples, the statistical analysis of the released rates was performed based on this compound. The model could demonstrate that release rates of eugenol from nanofibers were altered over time (Fhe tests . The aveLike many other essential oils, clove oil is widely used as herbal medicine and spice; they are generally recognized as safe for human health ; howeverC. pyri and C. pyricola females. We studied psyllid behavior in olfactometer choice tests and in the field. By conducting choice assays, we evaluated the relative preference or avoidance between single compounds and mixtures of clove essential oils for their ability to repel C. pyri and C. pyricola, and in altering host-seeking behavior in C. pyri. Experiments with this configuration which assess the number of insects avoiding treated plants in the olfactometer may be more predictive of insect behavior in the field. Furthermore, olfactometer assays separate the insect from contacting the odor source, specifically testing host-seeking behavior [C. pyri captures on traps equipped with M6 nanofibers with and without treatment (Our study demonstrated that a synthetic formulation of clove essential oil could be effective as a repellent to behavior ,52. Herebehavior . A true behavior . For thereatment ; this reColor is a factor that may play a more significant role in the psyllid’s behavior as VOCs on host finding. The repellent effect of eugenol could be counteracted by the presence of colored traps. Some psyllids’ and aphids’ chemotactic responses are enhanced when visual cues are present ,54,55. IC. pyri in olfactometer trials, the insect’s behavior was not disrupted in the open field, and it did not prevent the insect from finding and being caught in the trap. The olfactometer results suggest that eugenyl acetate has a synergistic effect with eugenol and β-caryophyllene in repelling of C. pyricola. Since the produced fibers did not contain these compounds, they would not be as effective against this species in the field. β-caryophyllene has already been described as a repellent against Diaphorina citri [C. pyri has been demonstrated in our tests, but not as a single compound; however, it could probably have a repellent effect in a dose-dependent matter.Nanofibers encapsulated the major compound of M6, eugenol; this was proven by the evaluation of release rates from new nanofibers, since eugenol was detected for 56 days . The secna citri . The repThis research demonstrates that nanofibers could be produced with biocompatible, biodegradable, non-toxic, and environmentally friendly polymers PCL/CA and solvents acetic acid and formic acid as described elsewhere ,60,61,62Further tests with higher amounts of the three active components of clove essential oil should be performed, as well as different distribution and a higher number of nanofiber materials in a given area to test the efficacy of the repellent in the field. Different fiber formulations with other polymer compositions could also be produced to stabilize the release rates of those compounds and enable the use of repellents to manage psyllids in the field. Our future goal is to develop an environmentally sound and cheap repellent emitting device with minimal side effects on non-target insect species in the orchard.