Infrared (IR) microlens arrays (MLA) have attracted increasing interest for use in infrared micro-optical devices and systems. However, the beam homogenization of IR laser light is relatively difficult to achieve because most materials absorb strongly in the IR wavelength band. In this paper, we present a new method for the application of double-sided quasi-periodic chalcogenide glass (ChG) MLAs to infrared laser homogenization systems. These are non-regular arrays of closely spaced MLAs. The double-sided MLAs were successfully prepared on the ChG surface using a single-pulse femtosecond laser-assisted chemical etching technique and a precision glass molding technique. More than two million close-packed microlenses on the ChG surface were successfully fabricated within 200 min. By taking advantage of ChG’s good optical performance and transmittance (60%) in the infrared wavelength band (1~11 μm), the homogenization of the IR beam was successfully achieved using the ChG quasi-periodic MLA. With the development of infrared technology, infrared lasers have been gradually applied in various fields, such as atmospheric monitoring , remote Booming applications in the IR region have created a demand for low-cost, highly efficient IR optics, but there have been few studies on infrared optics. Specifically, most materials have strong absorption in the infrared band, so the homogenization of IR lasers is more difficult . ChalcogHerein, an ultra-smooth homogenizer based on the ChG double-sided MLA was proposed for shaping an infrared pulse laser beam with a flat-topped profile intensity distribution. The technology is based on a single-pulse femtosecond laser-assisted chemical etching and precision glass molding process to achieve the rapid preparation of the double-sided MLA homogenizer. Within 180 min, about one million close-packed ChG microlenses with quasi-periodic distribution could be fabricated. Due to the high transmittance of ChG in the infrared band, we carried out the homogenization of laser beams with a wavelength of 1064 nm using the ChG double-sided MLA homogenizer. The results demonstrate the potential application of the fabricated double-sided ChG MLA for IR laser homogenization.w/v) hydrofluoric acid solution (HF) in an ultrasonic water bath . The physical properties of the ChG are shown in Using precision glass molding, the double-sided IR MLA was replicated using two quasi-periodic templates. A pre-dried piece of ChG was sandwiched between the two quasi-periodic templates, with the structured side of the template touching the ChG. The assembled module was then mounted in a high-temperature oven, as shown in The molding process was divided into four stages, as shown in By setting the scanning speed to 20 mm/s and the laser power to 5 mW, the quasi-periodic microlenses could be observed, showing an average size of 20 μm and a total area easily covering several square inches. The molding conditions were determined from a previous study, which examined the effect of temperature and pressure on the plasticity of ChG, as shown in 2/8h (n − 1), where D and h are the diameter and sag height of the microlens, respectively, and n is the reflectivity of the material. According to the formula, the focal lengths of the ChG microlenses and the BK7 MLA were 13.4 μm and 43.59 μm, respectively. The deformation ratio was ≈1% . The feature size of the ChG MLA fabricated was around 20 μm, so the scattering of wavelengths is expected at such a scale. The loss arose with wavelengths beyond 20 μm, as shown in The infrared imaging quality is the most basic optical characteristic of infrared microlenses. The infrared imaging capability of the quasi-periodic MLA was tested using an infrared microscope system, and the test result is shown in Beam homogenization is one of the most common applications for microlens arrays. Most microlens arrays are fabricated on polymer or plain glass, which are usually used for visible wavelengths. With a high transmittance for wavelengths, ranging from 1 to 11 µm, the ChG MLAs can be used for IR beam homogenization. The light beam homogenization was measured experimentally using the setup shown in After the homogenization of the ChG microlens array, the intensity distribution of the infrared laser in both modes spread to a flat-top distribution, shown in In summary, a large area of closely-packed, uniform double-sided quasi-periodic ChG MLAs were prepared using femtosecond laser wet etching combined with a precision molding technique and successfully applied to achieve the homogenization of infrared lasers. For mass production, this method shows great potential for preparing quasi-periodic closely-packed infrared microlenses. The focusing and imaging characteristics were tested, and the results showed that the ChG MLA has good infrared optical properties. Based on the good transmittance of the ChG MLA in the infrared band of 1~11 μm, the distribution of a 1064 nm semiconductor laser was homogenized from a Gaussian to a flat-top distribution. All the evidence proves that this large-scale infrared quasi-periodic MLA can be widely used in fields such as the night illumination of infrared lasers and infrared laser processing.


Safer prescribing in general practice may help to decrease preventable adverse drug events (ADE) and related hospitalisations.To test the effect of the Safer Prescribing and Care for the Elderly (SPACE) intervention on high-risk prescribing of non-steroidal anti-inflammatory drugs (NSAIDs) and/or antiplatelet medicines and related hospitalisations.A pragmatic cluster randomised controlled trial in general practice. Participants were patients at increased risk of ADEs from NSAIDs and/or antiplatelet medicines at baseline. SPACE comprises automated search to generate for each GP a list of patients with high-risk prescribing; pharmacist outreach to provide education and one-on-one review of list with GP; and automated letter inviting patients to seek medication review with their GP.The primary outcome was the difference in high-risk prescribing of NSAIDs and/or antiplatelet medicines at 6 months. Secondary outcomes were high-risk prescribing for gastrointestinal, renal, or cardiac ADEs separately, 12-month outcomes, and related ADE hospitalisations.Thirty-nine practices were recruited with 205 GPs and 191 593 patients, of which 21 877 (11.4%) were participants. Of the participants, 1479 (6.8%) had high-risk prescribing. High-risk prescribing improved in both groups at 6 and 12 months compared with baseline. At 6 months, there was no significant difference between groups  although SPACE improved more for gastrointestinal ADEs . At 12 months, the control group improved more . There was no significant difference for related hospitalisations.Further work is needed to identify scalable interventions that support safer prescribing in general practice. The use of automated search and feedback plus letter to patient warrants further exploration. It is not known how best to support safer prescribing in general practice. This trial tested a brief intervention comprising automated search, pharmacist delivered education and feedback, and automated letter to prompt patients to seek medicines review. The intervention had a partial, short-term effect. Brief interventions likely need repeating at regular intervals to achieve sustained improvement in general practice. Most prescribing of ongoing medication occurs in general practice, making safer prescribing in this context important for protecting patient safety. ADEs in primary care are common, and they cause distress and burden health systems. About one-fifth of ADEs in primary care may be preventable through safer prescribing.'Medication without harm' is a Global Patient Safety Challenge theme and top priority for the World Health Organization, which aims to reduce severe avoidable medication-related harm by 50% globally between 2017 and 2022. High-risk prescribing places patients at increased risk of ADEs, although it may be justified by the individual circumstances of the patient. People taking multiple medications are particularly at risk. Ethnic disparities for prescribing and ADEs exist. In New Zealand, Māori, and Pasifika people are more likely than other ethnic groups to be prescribed NSAIDs, and suffer associated ADEs at higher rates and younger ages.Cardiovascular medications (including anti-platelet medicines) and NSAIDs account for up to one-third of serious ADEs in older age groups. These include upper gastrointestinal bleeding, kidney injury, and exacerbation of heart failure, and so are an important target for improvement. One cluster randomised stepped wedge trial of a pharmacist-led intervention plus financial incentives found decreased high-risk prescribing of NSAIDs and related ADE hospital admissions; however, this trial did not have a true control group. Two recent systematic reviews found no high-quality evidence that interventions in general practice decrease ADEs or related hospitalisations, despite short-term improvements in prescribing.Trials of interventions to decrease high-risk prescribing in general practice have demonstrated improvements. The quality of evidence, however, is mixed, with many studies having short-term follow-up or interventions that are not readily scalable. Then, the effectiveness of SPACE on high-risk prescribing in general practice for NSAIDs and/or anti-platelet medicines and related ADE hospitalisations was tested.An intervention was developed to support safer prescribing in general practice, which can be applied to any prescribing topic (SPACE). A pilot study of SPACE showed promising results and good GP and patient acceptability. All GP practices in two regions of New Zealand were identified and eligible practices were invited via email and telephone to participate. Practices were not informed of the trial's high-risk prescribing topic. Eligibility was restricted to practices that used compatible electronic practice management systems and where all GPs consented to participate. Practices were excluded if they had participated in the SPACE pilot study or were participating in a non-trial initiative focusing on NSAID prescribing. Patients were included as participants if, at baseline, they were identified as at increased risk of gastrointestinal, renal, or cardiac ADEs from NSAIDs or anti-platelet medications  before extraction, written consent was not required from patients.A pragmatic cluster randomised controlled trial design was used with the practice as the unit of randomisation .20 Allications .20 WriEnrolled practices were stratified by region and practice size  and randomly assigned within strata to receive intervention or control in randomly occurring blocks of two and four. The random sequence was generated by a statistician not involved in recruitment or baseline data extraction. All outcomes and anonymised data extraction procedures were pre-specified and automated. Data at each time point were electronically extracted, de-identified, and sent by secure file to analysts , all of whom were masked to allocation until after trial completion.The intervention comprised:automated search of practice records using pre-defined algorithms to identify and generate for each GP a list of patients with high-risk prescribing for NSAIDs and/or anti-platelet medications;outreach visit from the trial clinical advisory pharmacist to provide a 1-hour group educational session with GPs on the prescribing topic;to meet one-on-one with each GP, and support them in reviewing their list of patients and selecting for each patient an intended action from a tick-box in the computer software ; andan automated letter from GPs to selected patients prompting patients to discuss their medicines at their next scheduled appointment.GPs were recompensed for their time with a NZ$100 gift voucher. All prescribing decisions were made as usual by GP and patient together. Control practices provided usual care.Practice prescribing data were extracted using automated pre-defined algorithms at baseline, and 6 months and 12 months after allocation. The primary outcome was the difference between SPACE and control practices in the proportion of participants with high-risk prescribing of NSAID and/or antiplatelet medicines at 6 months, after adjusting for baseline and clustering by practice . Seconda An average of 200 participants per practice and an intracluster correlation coefficient (ICC) of 4.68 × 10–7 for the primary outcome was estimated. Based on local feasibility study data, an 8% high-risk prescribing rate was assumed. Assuming approximately 12% of patients would be lost to follow-up over the 12-month study period, data from 8000 patients from 40 practices (20 practices in each group) with an average of 200 participants per practice were required to detect a statistically significant difference of 6% in the intervention group and 8% in the control group of high-risk prescribing at follow-up .The sample size calculation was based on previous trials demonstrating a clinically relevant 25–45% relative risk reduction in the proportion of high-risk NSAID or antiplatelet prescribing.Statistical analyses were performed according to the intention-to-treat (ITT) principle, with the use of mixed-effect models to account for clustering in the data. Primary analysis was by practice allocation. The proportions of participants with high-risk prescribing were analysed using random-effects logistic regression, with the individual as the unit of analysis and the practice included as the random effect to control for the effects of clustering. GLIMMIX with group-by-time interaction was used to assess the overall difference between intervention and control at 6 and 12 months. The model adjusted for the stratification factors including practice location (region A or B) and practice size .An inadvertent transcription error inverted the random group assignment of six practices before allocation  which was not apparent until the end of the trial. Analysis by allocation is presented in the Results section below. Per-protocol analysis and analysis by original random group assignment are provided in the online Supplementary Appendix.Of the 220 general practices identified, 110 fulfilled inclusion criteria, of which 39 (35.5%) agreed to participate and were enrolled between April 2018 and July 2019 . There wn = 1218/21 867 (5.6%) participants were lost to follow-up (n = 3215/21 867 (14.7%) participants.There were 191 442 patients registered in the 39 trial practices. Of these, 21 867 (11.4%) were identified as participants (at increased risk of ADEs from NSAIDs or antiplatelet medications), of whom 1479 (6.8%) had high-risk prescribing over the previous 14 weeks . Demograollow-up . By 12 mn = 254/828 (31%) participants with high-risk prescribing at baseline. Poor engagement resulted in the trial pharmacist having one-on-one sessions with only 70% of GPs and reviewing only 416 of the 683 participants (61%) identified at intervention delivery as having high-risk prescribing (Supplementary Table S1).There were often delays between baseline measures, group education, and one-on-one list review — ranging from 1 week to 6 months — mostly because GPs were 'too busy'. Often, the automated search was re-run to generate an up-to-date list of patients with high-risk prescribing for one-on-one review. In three intervention practices (two large and one medium), there was minimal or no engagement with the intervention (Supplementary Table S1); one practice did not receive the group education, and only one of 15 GPs agreed to meet with the pharmacist for the one-on-one list review. These three intervention practices included n = 97, 23%); ‘No letter but review’ ; and ‘No action’ . The reasons for ‘No letter but review’ included GP concern about upsetting the patient; patient not understanding written English; patient due to be seen soon; or GP preference to leave themselves a note. The reasons for ‘No action’ were mostly that the high-risk prescribing had already ceased ; the patient had moved practice; the GP considered the individual circumstances of the patient justified the high-risk prescribing; or the GP did not agree that the prescribing was high-risk (Supplementary Table S1).The GP tick-box selections for the 416 participants reviewed were ‘Letter’ , although high-risk prescribing for gastrointestinal ADEs had improved significantly more in the SPACE group , which was sustained at 12 months  (Supplementary Table S2).Analysis by original random group assignment showed the SPACE group was significantly more likely to have high-risk prescribing than the control group at 6 months  (Supplementary Table S3).There was no significant difference between the groups for total hospitalisations or for related gastrointestinal, renal, or cardiac ADE hospitalisations in the 12 months following baseline adjusting for the 12 months before baseline  while improving prescribing for renal and cardiac ADEs requires stopping medication, which GPs can find more challenging. A further limitation was that some practices enrolled in the contemporaneous prescribing initiative during follow-up, which confounded the results but reflects the real-world conditions in which the study was conducted. A further limitation was the transcription error, where one block of six practices were allocated to the inverse of the random group assignment generated. ITT analysis is presented by original random group assignment in the appendices, although this analysis risks a type two error (introducing bias towards the null hypothesis).The strengths of this study include pragmatic trial design, and a scalable intervention for use in general practice that may be applied to any prescribing topic, which includes novel elements of pharmacist supporting GPs to review a list of patients and select some for an automated letter prompting them to seek medicines review. Important limitations include lower than expected engagement and uptake of the intervention. For one-third of SPACE participants, practices did not engage or there was minimal engagement, making it difficult to show an effect. For practices that did engage, it was often difficult for the trial pharmacist to secure time with GPs, delaying intervention delivery. Unlike in the SPACE pilot study, the trial pharmacist was not known to practices and GPs which may explain the difficulties. Complex interventions combining educational outreach visits, audit, and feedback can work, as can interventions involving pharmacists in general practices, especially when combined with financial incentives. Interventions that include empowering patients have also shown promise. An intervention that included seven prescribing audits, seven face-to-face or group sessions, and then sessions every 4 months showed a lasting effect on high-risk prescribing.This study's findings are consistent with previous research investigating ways to improve prescribing in general practice, which had limited success.Further work is needed to identify scalable interventions that support safer prescribing in general practice in the longer term. The role of practice-based clinical advisory pharmacists and the use of automated searches to generate lists of at-risk patients for each GP and letters to patients to prompt medication review warrant further exploration.


BackgroundAlthough evidence has emerged indicating that patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia present a high risk of venous thromboembolism (VTE), its real incidence and best diagnosis course remain unclear. In this study, we aimed to determine the incidence of pulmonary embolism in these patients and the role of D-dimer serum level as a predictive factor of a new VTE event.MethodologyThis was a single-center retrospective observational cohort study conducted in a tertiary hospital. All patients admitted to the infectious diseases ward with SARS-CoV-2 pneumonia with clinical or laboratory criteria for suspected VTE events were eligible for inclusion in the study. The t-test or Mann-Whitney U test was used to analyze the differences between the with-VTE group and the without-VTE group.ResultsOverall, VTE incidence was registered to be 30%. Chest computed tomography angiography data revealed thrombus mainly in segmental  and subsegmental pulmonary artery branches . No thrombus on major branches was documented. D-dimer serum levels  were analyzed, and, despite a consistent tendency of higher values in the with-VTE group, no statistical difference was observed. Moreover, no statistical difference was observed between the two groups in mortality rates.ConclusionsA clear higher risk of VTE events in SARS-CoV-2 pneumonia patients was not documented, and a link between the impact of VTE occurrence and a worse prognosis was not demonstrated. Therefore, we suggest that the use of D-dimer serum level should not be used as a predictor of VTE in SARS-CoV-2 pneumonia patients. Since its initial identification in December 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide and has been declared a pandemic by the World Health Organization. Although not primarily a thrombotic process, inflammation and hypoxia with acute lung injury are known mechanisms that lead to a profound inflammatory state due to cytokine production, macrophage, and endothelial activation-related processes associated with excessive inflammation and diffuse intravascular coagulation ,4.Furthermore, SARS-CoV-2 patients usually present with markedly elevated serum levels of D-dimer with slight or no changes in partial thromboplastin time or activated partial thromboplastin time. This profile further complicates its use as an indicator of a new intercurrent VTE because elevations in D-dimer are very common in these patients; moreover, it has low specificity for VTE events .In this study, we aimed to determine the incidence and risk factors of VTE in SARS-CoV-2 patients, specifically pulmonary embolism, admitted to hospital care in a medical ward. In addition, we aimed to clarify the relevance of D-dimer serum level as an indicator of a new VTE event in these patients.Study design and populationThis single-center, retrospective, observational cohort study was conducted in a tertiary hospital in Lisbon, Portugal. Data were collected from consecutive patients admitted to the infecciology hospital ward from March 1 to November 31, 2020.Eligibility criteria included patients aged 18 or more and admitted to the infecciology hospital ward with a clinical and microbiological diagnosis of SARS-CoV-2 pneumonia. This diagnosis was made only when all the following criteria were fulfilled: (1) Confirmation of SARS-CoV-2 infection by a reverse-transcriptase polymerase chain reaction test on a nose/throat swab or a sputum sample positive for the virus; (2) clinical symptoms consistent with the diagnostic criteria of acute pneumonia ; (3) Radiological confirmation of lung involvement either by chest X-ray or chest computed tomography; and (4) fulfillment of Berlins’ acute respiratory distress syndrome (ARDS) criteria.Patients were eligible for study inclusion if either a clinical  or laboratory (variation of D-dimer serum levels of at least 10% within 48 hours) criteria of a suspected VTE event were verified during the hospital stay. These patients were submitted to chest computed tomography angiography and were further divided into two groups according to the clinical and laboratory findings: the with-VTE group or the without-VTE group. All patients with documented VTE events received anticoagulation therapy after VTE diagnosis. Thrombotic venous disease prophylaxis with low-molecular-weight heparin, as part of the treatment protocol, was administered according to the patients’ individual risk, after individual clinical evaluation, at admission.Exclusion criteria included age less than 18 years, patients with incomplete data, and patients with a major contraindication to anticoagulation therapy prior to the diagnosis of pulmonary thromboembolism.The Ethics Committee and Institutional Review Boards approved the study protocol as minimal-risk research, using data collected for routine clinical practice, and waived the requirement for informed consent (CES 970/2020).Data collectionPatient demographic characteristics were recorded at baseline for all patients. For those with clinical or laboratory suspicion for a VTE event, data were recorded on comorbidities and VTE risk factors.Clinical and laboratory data, including Wells score, anticoagulation therapy, and remdesivir and dexamethasone therapy, were also obtained for all selected patients at three time periods: at patients’ admission, 48 hours before VTE suspicion, and on the day of VTE suspicion. Patients with clinical or laboratory suspicion for a VTE event were submitted to chest computed tomography angiography, and its results and Pulmonary Embolism Severity Index (PESI) score were collected. Patients were followed until discharge or death, with hemorrhagic events (defined as any event of documented hemorrhage that motivated suspension of anticoagulation therapy) and clinical outcomes registered for further analysis.OutcomesThe primary outcome of our study was the incidence of VTE, specifically pulmonary embolism, among patients admitted to the infecciology hospital ward with SARS-CoV-2 pneumonia with clinical or laboratory VTE suspicion. The secondary endpoints were the analysis of the influence of a VTE event on clinical outcome (discharge vs. death) and the determination of the relevance of D-dimer level as an indicator of a new VTE event.Statistical analysisWe planned to include a total of 400 patients considering a predicted incidence rate of VTE events of 10%. Quantitative variables were expressed as medians (interquartile range) and categorical variables were expressed as numbers (percentage). The t-test or Mann-Whitney U test was used to analyze the differences between the with-VTE and without-VTE groups. Statistical analysis was conducted using SPSS software version 21.0 .In total, 370 patients were included in this study . Of this, 23 patients met all the inclusion criteria and underwent chest computed tomography angiography due to clinical or laboratory suspicion of a VTE event  and included 13 (56.5%) males, as presented in Table Incidence of venous thromboembolism and its predictorsVTE events were observed in seven patients with an incidence of 1.9% (7 in 370 patients) during the study. In the selected group with suspected VTE events, submitted to analysis, VTE events were observed in 30% of patients (7 in 23 patients).The mean PESI score in patients with VTE events was 97.29 (±19.9), and chest computed tomography angiography revealed the presence of pulmonary artery thrombus mainly in segmental  and subsegmental branches . In none of the observed patients pulmonary artery thrombus on major branches was observed  and Wells criteria for pulmonary embolism score was not statistically different between the with-VTE and without-VTE groups. The mean arterial pressure at the time of suspected VTE, hemoglobin level, and C-reactive protein were also not different between the groups. To account for possible confounding factors for VTE events, the number of patients on remdesivir, dexamethasone, and anticoagulation therapies were evaluated in the with-VTE group and were not statistically different for those observed in the without-VTE group Table .4) ng/mL; without-VTE group: 9,342 (±1.6 × 104) ng/mL; p = 0.25) , and its low repercussion on patients’ clinical state and organ dysfunction (no unstable patients were registered at the date of suspected VTE event or posterior need of ICU care), even when superimposed with a SARS-CoV-2 infection, which usually dictates an extensive pulmonary commitment.The impact of specific VTE events  in these patients should, therefore, prompt an important clinical discussion, considering the relevance of its presence in the patients’ outcomes, and, especially, the severe hemorrhagic complications, secondary to anticoagulation therapy, observed in this subset of patients.Our study presents some strengths, such as the systematic inclusion of patients admitted to the hospital without any selection bias, rigorous determination of laboratory D-dimer serum levels for all included patients for analysis, and continuous follow-up of all included patients until the outcome was verified. However, some limitations of our study should be acknowledged. First, it is a retrospective, single-center, small sample study. The calculated population initially idealized was not reached, reducing its statistical strength to generalize the obtained results and sustain further conclusions. Hence, the study findings need to be confirmed by larger studies. Second, in regards to VTE event characterization, venous Doppler ultrasonography of the lower limbs was not done in all patients, restricting the statistical analysis to the occurrence of pulmonary embolism and reducing the ability to identify other VTE events, such as deep vein thrombosis.We hope that our study may contribute to improving our understanding of VTE risk in SARS-CoV-2 pneumonia patients and contribute to better hospital care for these patients.Our study has not demonstrated a clear higher risk of VTE events in SARS-CoV-2 pneumonia patients or an impact of VTE occurrence in patient prognosis. VTE events were registered in a minority of patients studied, and, in those patients, the thrombotic disease was restricted to segmental and subsegmental branches, with no cases of thrombus in major pulmonary artery branches. Furthermore, the collected data did not show any association between VTE events and increased mortality, possibly indicating a low clinical impact of VTE events.Additionally, D-dimer serum levels, or its variation along the course of the SARS-CoV-2 infection, failed to demonstrate a good predictive accuracy of new VTE events in this specific set of patients. Therefore, this study suggests that the suspicion of a VTE event should rely on the patient’s clinical evaluation, over the sole use of D-dimer serum levels, and should be confirmed with computed tomography pulmonary angiography. Although more prospective and randomized studies are clearly necessary, this study does not support the use of this marker as a sole predictor of VTE events in the clinical diagnosis and decision-making among coronavirus disease 2019 patients.


Co-existence of multiple chronic diseases is increasingly becoming a norm among ageing population. The study aims to investigate the prevalence of multimorbidity and the association between anthropometric measures of obesity and multimorbidity among men and women aged 60 years and above in India.The present study is based on the first wave of the Longitudinal Aging Study in India. The analytical sample size for the study was 28,050 older adults aged 60 years and above. Descriptive statistics and multivariable analysis using logistic regression models were conducted.Body Mass Index (BMI) based-obesity is more prevalent among older women than men (26.3% vs. 17.6%). Similarly, higher proportion of older women was at high-risk waist circumference (37.1% vs 8.9%) and waist-hip ratio (78.5 vs 75.4%) than men respectively. In Model-I, after controlling for several covariates, older adults with overweight/obesity were 1.6 times more likely to have multi-morbidity than non-obese older adults . Similarly, older adults with high-risk waist circumference  and waist-hip ratio  also had higher odds of having multi-morbidity in reference to their counterparts. In model-3 it was found that females with high-risk waist-hip ratio had 14% lower odds of multimorbidity than males with high-risk waist-hip ratio .The findings of the study show significant gender difference in the prevalence of multimorbidity, men being at increased risk in the multivariate analysis which is uncommon in the existing epidemiological research. Interactive effect of male gender with anthropometric measures on multimorbidity reported in our study probably due to increased unhealthy behaviours among men requires further research. According to the Global Burden of Disease 2015 study, higher body mass index (BMI) is one of the highest and increasing risk factor contributing to poor health conditions . A higheMulti-morbidity at older ages leads to low quality of life, higher mortality rates, low physical and mental health, cognitive decline and higher healthcare expenditure , 7. In aCo-existence of multiple chronic diseases is increasingly becoming a norm among ageing population . On the The present study is based on the first wave of the Longitudinal Aging Study in India (LASI) conducted during 2017–18 . The LASThe LASI survey is conceptually comparable to the United States Health and Retirement Study (HRS) and other HRS-type surveys in various countries, including China  and England . Along with its uniqueness of comparability with studies in other countries, LASI also considered features unique to India, including its institutional and cultural characteristics. LASI is conducted through a partnership of the International Institute of Population Sciences (IIPS), Harvard University, and the RAND Corporation . AdditioMulti-morbidity refers to the presence of two or more chronic diseases , 27–30 wThe variables controlled for the present study were taken into consideration after extensive literature review. Body Mass Index (BMI) was calculated as weight in kilograms divided by height in meters squared. The respondents having body mass index of 25 and above were categorized as obese/overweight . OverweiAge was categorized as young old (60–69 years), old-old (70–79 years) and oldest-old (80 + years). Education was categorized as no education/primary schooling not completed, primary completed, secondary completed and higher and above. Marital status was categorized as currently married, widowed and others (separated/never married/divorced). Working status was categorized as working, not working/retired, and never worked. Tobacco and alcohol consumption was assessed through the questions ‘have you ever smoked tobacco or used tobacco products?’, ‘And have you ever consumed alcoholic beverages such beer, wine, liquor, etc.?’. It was coded as no and yes . PhysicaThe monthly per capita consumption expenditure (MPCE) quintile was measured using the information related to household-level consumption of food and non-food items. The reference periods for food expenditure were seven days and for non-food expenditure were 30 days and 365 days. These expenditures have been standardized to the 30-day reference period. The MPCE is computed and used as the summary measure of consumption  and diviDescriptive statistics along with bivariate analysis was presented in the paper. The analysis was stratified by gender. To signify the association between gender differentials, proportion test  was usedThe binary logistic regression model is usually put into a more compact form as follows:The parameter ble used .The multivariable analysis had four models to explain the adjusted estimates. Model-1 provides the adjusted estimates for the control variables. Model-2, model-3 and model-4 provide the interaction effects , 44 for Table Table Table The present study based on large country-representative survey information of older Indian adults aged 60 years and above, has shown the associations between several anthropometric measures and multi-morbidity in older ages with a special focus on gender difference in such associations. Although bivariate analyses in the current study showed female disadvantage in the prevalence of multi-morbidity, higher likelihood of multi-morbidity among older men than women in the multivariate analysis after adjusting for potential confounders was contrary to earlier studies in developed as well as developing countries that have revealed greater odds of multi-morbidity among older women, relating to their longer life expectancy and poor health status compared to older men , 45–48. Older adults who were measured as obese in the study population, consistent with a couple of previous studies, were 61% more likely to report having multi-morbidity than older adults with no obesity , 22, 47.The current analysis shows that oldest age group is at reduced risk for multi-morbidity and is consistent with earlier studies , 53. ThiIn agreement with a few cross-sectional as well as longitudinal studies, in the present study, late-life physical inactivity was associated with a higher likelihood of multi-morbidity , 62–64. Furthermore, the interactive effect of gender in the associations of obesity-related measures and multi-morbidity shows that older men with overweight/obesity are at greater risk for multi-morbidity than their women counterparts. The male disadvantage in being multi-morbid observed in the current analyses can be attributed to the hormonal differences between males and females, genetic factors and differences in clinical severity .. SimilaFinally, the regional variations and urban- rural gradient in the prevalence of multi-morbidity suggest that older adults from southern states of the country which are socioeconomically advantaged with relatively developed healthcare system and higher levels of education and income , and thoThere were some limitations to be acknowledged in the present study. The cross-sectionality of the study makes it unfeasible to infer the causation in the observed directions of the relationships. Also, it uses a relatively simple definition of counting diseases for measuring multi-morbidity in variance with earlier studies , 73, 74.The results of the study show significant gender differences in the prevalence of multi-morbidity between older men and women, men being at increased risk in the multivariate analysis which is uncommon in the existing epidemiological research. The findings highlight the need for better management of chronic conditions in older adults in primary care, increased prevention measures including nutritional interventions, physical activity promotion etc., information and physician education with a special focus on those who are obese, overweight or with high-risk waist circumference and waist-hip ratio. Interactive effect of male gender with anthropometric measures on multi-morbidity reported in our study probably due to increased unhealthy behaviours among men, requires further research. Also, studies are required on multi-morbidity patterns and several combinations of morbidities and its impact stratified by gender among the aging population in India.


Management of acute coronary syndrome (ACS) has emerged as a challenge during the COVID-19 era. There has been a significant increase in the morbidity and mortality associated with ACS both as a direct and an indirect consequence of the pandemic. In this review, we provide an overview of the impact of COVID-19 on patients presenting with ACS and current practices for managing patients presenting with chest pain during the pandemic and for ensuring safety of healthcare professionals. We also discuss treatment strategies and post-ACS care along with current and future perspectives for management of ACS during future waves of COVID-19 infection or similar pandemics. However, a significant number of patients have presented with late-stage cardiac diseases, such as late-presenting ACS, and with serious cardiac complications including cardiac arrests and out-of-hospital death.5 With improved awareness of the disease and public messaging, subsequent COVID-19 waves have not correlated with a similar decline in acute myocardial infarction-related hospitalizations.6 The pandemic has further given rise to an immense demand for resources, leading to diversion and deferral of essential preventive and elective medical services for patients with stable cardiac problems.7The coronavirus 2019 (COVID-19) pandemic has significantly impacted management of cardiovascular emergencies across the world. Early in the pandemic, an unforeseen decline in hospital admissions of patients with acute cardiovascular emergencies was noted.8 Managing late-presenting myocardial infarction (MI) has always been challenging and complex, and COVID-19 has added another layer of complexity. In the US, a statistically significant increase in deaths due to ACS has been recorded during the pandemic, correlating geographically with the number of COVID-19 cases in the area.7 Early in the pandemic, prospective data from 55 international centers was used to create a “COVID-ACS” registry that included patients who were either COVID-19 positive or had a high index of clinical suspicion for the infection. Analysis of registry data showed that mortality and complications were significantly higher in COVID-ACS patients compared to the same population in the pre-pandemic era .9 For this reason, it is important to address ACS in patients with COVID-19 differently to improve morbidity and mortality while protecting the treatment staff.Increased morbidity and mortality were noted secondary to ACS and were thought to be related to a significantly longer time from symptom onset to hospital presentation in addition to the delay in performing primary percutaneous coronary interventions (PPCI).10 Thus, any patient presenting to the emergency department or clinic should be considered as possibly infected with COVID-19, and a surgical mask should be used as a bare minimum layer of protection when examining the patient.12 Patients with a high possibility of or known COVID-19 infection should be examined using adequate personal protective equipment (PPE), which includes N95 respirators and disposable gowns and gloves for standard airborne and contact precautions.12 Patients should also wear a mask during all encounters, especially because they can be contagious during the disease’s incubation period.Effectively triaging patients presenting with cardiopulmonary symptoms has become challenging during the COVID-19 pandemic. Healthcare workers in cardiac catheterization laboratories managing patients presumed to be infected with COVID-19 have higher risks of infection than those managing patients with low risk of infection (2.3% vs < 0.25%).13 Simultaneous assessment of potential COVID-19 infection should begin at the point of first contact because it can protect the care team and determine the strategy for managing ACS. Currently, hospitals are testing all admitted patients for COVID-19 to optimize care and protect healthcare workers.A high index of suspicion for ACS should be maintained for any patient presenting with symptoms of angina or angina equivalent. In addition to a good history and physical examination, the treatment team should evaluate patients using point-of-care ultrasound examination, electrocardiogram (EKG), and cardiac biomarkers.14 The same association was observed with the increase in B-type natriuretic peptide.15 It is important to differentiate a type 1 MI from a supply-demand mismatch , immune- and inflammatory-mediated myocarditis, pulmonary embolism, or stress-induced cardiomyopathy in the setting of COVID-19 infection.16 A case series from New York City in the beginning of the pandemic showed that, out of 18 patients infected with COVID-19 who developed ST-segment elevations on the EKG, 10 patients were found to have noncoronary myocardial injury.17 Subsequently, in a large registry of patients diagnosed with COVID-19 infection and presenting with typical or atypical symptoms consistent with ACS, and an EKG finding of ST-segment elevation, no culprit vessel lesion was found on angiography in 23% of patients (41 of 179); this was significantly higher than a finding of 1% of patients (5 of 459) in a similar matched control group from the pre–COVID-19 era.18 A history, troponin trend, serial EKGs, and other noninvasive imaging modalities can be helpful to establish a diagnosis. Point-of-care ultrasound is valuable in this population to help differentiate a STEMI from STEMI “mimickers,” which are more commonly encountered in patients with COVID-19 pneumonia. The administration of thrombolytic agents in the latter population could be catastrophic and cause significant harm to such patients.19It is important to note that rising cardiac biomarkers in the setting of COVID-19 infection may be due not only to acute plaque rupture or erosion leading to ACS but also to direct myocardial injury, such as myocarditis. An increase in troponin is 13 times more common in severe COVID-19 infection than in milder presentations and has prognostic value.21 In addition, a recent randomized controlled trial confirmed that there was no difference in short-term outcomes for patients with mild-to-moderate COVID-19 symptoms, regardless of whether such medications were continued or discontinued.22Once ACS is suspected, medical therapy should be instituted immediately along with a decision whether to proceed with an invasive strategy. Medical therapy for ACS in COVID-19 patients is identical to patients without COVID-19. This includes dual antiplatelet therapy (aspirin and a P2Y12 inhibitor), intravenous or subcutaneous anticoagulation, statins, and beta blockers (if no contraindications). Oxygen is only administered in the setting of hypoxia. Reports from the early pandemic phase showed that angiotensin converting enzyme inhibitors and angiotensin-receptor blockers might be harmful, but further investigations negated these findings.23 PPCI is always preferred over fibrinolysis whenever adequate PCI facilities are available, and this should continue to remain the standard of care in patients presenting with STEMI with a low suspicion of COVID-19. Because there is a significant mortality benefit in patients receiving PPCI over intensive pharmacological therapies, especially in cases of anterior wall MI and concurrent cardiogenic shock, PPCI should still be the preferred approach for patients with high suspicion of COVID-19 presenting with cardiogenic shock and anterior STEMI.There is strong evidence supporting the superiority of PPCI over fibrinolysis for reducing short-term complications and mortality in patients presenting with STEMI.24Providing necessary PPE and training the catheterization laboratory staff in its correct use could decrease the spread of infection during the procedure. Moreover, using dedicated rooms for patients with known or presumptive positive COVID-19 infection and limiting the number of healthcare workers to only those who are essential for procedure support can further help control in-hospital spread of infections.25It is important to note that COVID-19 patients are hypercoagulable; therefore, the healthcare team must pay particular attention to maintaining activated clotting time in the target range while performing PPCI. In a single-center study including 115 consecutive STEMI patients undergoing PCI, COVID-STEMI patients had higher rates of multivessel thrombosis with higher need for aspiration thrombectomy and glycoprotein IIb/IIIa use, higher inpatient mortality, greater rates of ICU admission, and longer length of stay compared with non-COVID STEMI patients treated during the same period.18 Furthermore, patients with COVID-19 had higher average door-to-balloon times . This was associated with higher rates of in-hospital deaths, recurrent MIs, and repeat unplanned revascularization in the group diagnosed with COVID-19 compared with controls .18The North American COVID-19 Myocardial Infarction (NACMI) registry was created by the SCAI, American College of Cardiology, and Canadian Association of Interventional Cardiologists to fill the knowledge gap regarding outcomes in patients with diagnosed COVID-19 presenting with STEMI. Results from this prospective observational registry from 64 US and Canadian sites showed that only 78% of the patients with diagnosed COVID-19 infection who presented with STEMI received an angiogram compared to > 99% in the control group, and a higher number of patients received medical therapy (20% vs 2%).10 Using the best available data to estimate risk of mortality from STEMI and staff infection risk at that time, the authors found that for the majority of patients, PPCI offers mortality benefit over fibrinolysis. However, in those with severe COVID-19 and nonanterior STEMI without the presence of cardiogenic shock, the mortality benefit of PPCI was minimal and fibrinolytic therapy could be considered to reduce provider risk of COVID-19 infection. Given the highly efficacious and safe vaccines against COVID-19, this important ethical question and its implications may be significantly diminished, especially in health systems where all staff wear PPE and PPCI remains the standard of care for STEMI even in patients with COVID-19.Before COVID-19 vaccines became available, there were concerns regarding balancing risk of staff exposure during PPCI in a COVID-19–positive patient and risk of mortality and morbidity from a STEMI patient treated conservatively or with fibrinolytics alone. A decision-analysis framework was performed to evaluate this tradeoff.26 Another concern with fibrinolysis is risk of bleeding in STEMI mimickers, such as myocarditis. Hence, PPCI with adequate precautions remains the standard of care in STEMI patients with COVID-19. A fibrinolysis-based strategy may be considered at non-PCI capable referral hospitals or in specific situations where primary PCI is deemed inappropriate.Fibrinolysis in patients presenting with STEMI in the setting of pneumonia has been independently associated with increased in-hospital mortality, especially among patients aged > 60 years.For patients presenting with symptoms concerning for ACS with elevated troponin and no ST-segment elevation, COVID-19 screening with highly accurate reverse transcription polymerase chain reaction should be performed as soon as possible. Risk can be assessed using traditional scoring systems, such as Thrombolysis in Myocardial Infarction (TIMI) and Global Registry of Acute Coronary Events (GRACE) scores, and serial EKGs, troponin, and clinical monitoring should be performed. If there are no urgent indications for revascularization, PCI can be deferred until the patient’s infectivity status is ascertained.27 Thus, efforts should be made to reduce the hospital stay whenever possible. A common practice after PCI involves admitting all patients to critical care units for close observation, which frequently leads to a longer hospital course.28 To reduce length of hospital stay, the patient’s risk of contracting in-hospital COVID-19 infection, and utilization of critical care beds and resources, it is helpful to stratify risk for complications after STEMI revascularization using validated risk-predicting scores, such as CADILLAC or Zwolle risk scores, and triage patients based on risk of post-STEMI complications.30 The pandemic has made it necessary to increase emphasis on the discharge planning process to prevent recurrent ACS and avoid hospital readmission, thereby reducing the risk of in-hospital COVID-19 infection.31 Close follow-up with an outpatient cardiologist, either in office or by telemedicine, should be arranged before discharge.Hospitalization remains one of the risk factors for COVID-19 infection.32 However, there is significant vaccination hesitancy recorded in different parts of the country, and this—along with new emerging strains like the Delta, Delta-plus, Omicron, and Lambda variants—continues to pose risks for a wave of milder COVID-19 infections in the near future.33 The factors that resulted in increased complications and mortality from ACS in past waves will continue to persist in future waves of milder disease. It is important for healthcare systems to evolve and create policies to ensure that treatment is not delayed and standard of care is maintained for patients presenting with ACS in future similar circumstances. The use of telemedicine played a major role in providing healthcare services during the pandemic.34 The effectiveness of telemedicine for preventive cardiology and post-ACS care should be studied further since it could result in regular healthcare follow-up without visiting a cardiologist’s office.COVID-19 vaccinations have shown to be safe and effective in preventing cases and reducing the severity of the disease, hospitalization, and death.The COVID-19 pandemic has directly and indirectly impacted the care of patients with cardiovascular emergencies. COVID-19–positive patients with acute coronary syndrome (ACS) present late and have higher incidences of cardiogenic shock and in-hospital mortality compared with the pre–COVID-19 ACS population.Primary percutaneous coronary intervention with full personal protective equipment and adequate protection in the catheterization lab remains the mainstay for patients presenting with STEMI and confirmed or a high index of suspicion for COVID-19 infection.Efforts are needed to increase public health messaging and education regarding avoiding delays and seeking immediate medical care for suspected acute cardiac conditions and to increase vaccination against COVID-19, which has been proven to be safe and effective.


IntroductionNeglected clubfoot comprises clubfoot deformities with late presentation and weight-bearing on the affected foot. These deformities are stiff and need an aggressive approach for cast-based management. The modified versions of the Ponseti technique have been found effective in treating these deformities. However, these techniques’ long-term outcomes in neglected clubfoot and related correction maintenance with time progression have not been investigated. The current study aims to analyze the changes in deformity correction after a minimum of two years of follow-up in neglected clubfeet treated with a modified Ponseti method of corrective casting.MethodsWe retrospectively analyzed the clinical records of 25 patients with 38 neglected clubfeet with a mean follow-up of 37.9±4.1 months after the initial correction of the deformity. These patients were managed with a modified Ponseti technique. The modified technique incorporated simultaneous deformity manipulation before equinus correction and additional dorsiflexion manipulation after two weeks of tendo-achilles tenotomy. The Pirani and Dimeglio scores and individual deformity corrections at the final follow-up were compared with those at final cast removal.ResultsNo significant differences were observed between the initial and the final follow-up Pirani and Dimeglio scores. Concerning the deformity correction parameters, there was a significant loss in heel varus and foot abduction correction. However, the change in these parameters was small (less than 3 degrees).ConclusionThe neglected clubfoot deformity can be satisfactorily corrected with an aggressive Ponseti based corrective manipulation and casting at a low relapse rate. However, minor loss of deformity correction is noticed after two years of treatment which is not reflected in clinical scores. Therefore, a strict long-term follow-up and careful deformity assessment are required to predict the recurrence in these cases. Neglected clubfoot comprises the clubfoot deformities that have not been adequately managed before the walking stage . The preConsidering these shortcomings, some authors investigated the role of corrective manipulation and casting in the management of neglected clubfoot -6. They We retrospectively reviewed the neglected clubfeet patients presented in a tertiary care center for two years. The inclusion criteria were: clubfoot deformity that had received inadequate/nil treatment before walking stage, minimum age of 2 years at presentation, a minimum follow-up of two years, availability of clinical records of individual deformity correction , and Pirani scores and Dimeglio scores at the final cast removal. In addition, we excluded the patients who were non-compliant with the post-correction bracing protocol during the follow-up. The foot manipulation in these cases was performed using a modified version of the Ponseti technique described by Mehtani et al. . FollowiIndividual deformities, Pirani scores , and DimA total of 25 patients  with 38 club feet  were reviewed. The mean age was 3.3±1.6 ( range 2-7) years. The mean follow-up was 37.9±4.1 (range 30-44) months. No significant differences were observed between the initial and the final follow-up Pirani and Dimeglio scores (Table The Ponseti casting technique is currently the standard management of clubfoot. The technique is effective in late presenting clubfeet also -13. HoweFew studies have emphasized the role of corrective casting and manipulation in neglected clubfoot -13. The There are some limitations of the current analysis. First, this was a retrospective review of clinical records. Therefore, the impact of factors like severity of the deformity, age, gait patterns of the deformed foot, orthopedicians expertise, number of casts applied, patient compliance to the bracing protocol before the follow-up could not be analyzed. Second, the study analyzed the outcomes of single center-based findings, and those may be extrapolatable to a larger population. Third, it can’t be predicted which feet will require more prolonged casting or require a short casting period. Fourth, the findings of this study can’t predict the effectiveness of cast-based treatment in older patients with neglected clubfoot. Fifth, the influence of previous inadequate management on deformity correction can not be predicted. Last, the study predicts deformity changes at a mean follow-up of approximately 3 years. Thus, it is difficult to state whether these changes were temporary, persistent, progressive, or nonprogressive. Despite these limitations, the current analysis highlights the effectiveness of cast-based methods in neglected clubfoot and the need for a strict long-term follow-up protocol to predict early recurrence.Modified Ponseti technique-based casting is an effective method of correction of neglected clubfoot. The deformities can be satisfactorily corrected with an aggressive corrective manipulation and casting at a low relapse rate. There is a minor loss of deformity correction after two years of treatment but that is not reflected in the clinical scores. Therefore, a strict long-term follow-up and careful deformity assessment are required to predict the recurrence in these cases. Further, long-term sequelae of neglected clubfoot need to be investigated to understand the prognosis of these deformities.


The consumption of tobacco products is regarded as the largest preventable risk factor for a large number of serious diseases. It causes around 121,000 deaths per year in Germany alone , 4. SmokThe analyses presented here are based on self-reports on current smoking  and on the age of smoking initiation. The data is taken from the KiGGS cohort, a study that follows the participants of the KiGGS baseline study (2003-2006)  into aduThe German Health Interview and Examination Survey for Children and AdolescentsData owner: Robert Koch InstituteAim: Providing reliable information on health status, health-related behaviour, living conditions, protective and risk factors, and health care among children, adolescents and young adults living in Germany, with the possibility of trend and longitudinal analysesStudy design: Combined cross-sectional and cohort studyKiGGS survey waves►KiGGS baseline study (2003-2006), examination and interview survey►KiGGS Wave 1 (2009-2012), interview survey►KiGGS Wave 2 (2014-2017), examination and interview surveyKiGGS cross-sectional studyPopulation: Children and adolescents with permanent residence in GermanyAge range: 0-17 yearsKiGGS cohort studySampling: Re-invitation of everyone who took part in the KiGGS baseline study  and who was willing to participate in a follow-upAge range KiGGS Wave 1: 6-24 years Age range KiGGS Wave 2: 10-31 years www.kiggs-studie.de/englishMore information is available at The proportion of people in the sample who smoked increased from 32% to 43% between the periods during which the KiGGS baseline study and KiGGS Wave 1 were undertaken. 85% of adolescents who smoked and 78% of non-smoking adolescents did not change their smoking behaviour in young adulthood. Whereas 15% of young people who smoked quit smoking between the two survey periods, 22% of non-smokers starting smoking during this time. The analyses of data covering the age when a respondent began to smoke show that almost nine out of ten participants who ever smoked started smoking before the age of 18.Although no differences were found between the proportions of girls and boys who smoked during adolescence, the proportion of smoking in young adulthood increases more considerable in young men than in women. The main reason for this difference is that women who smoked as adolescents quit smoking significantly more often than men (19% vs. 9%) during the transition into adulthood. At the same time, formerly non-smoking male adolescents have started smoking more often; however, this difference is not statistically significant .Finally, there is a strong correlation between SES and smoking behaviour. For both survey periods, a larger proportion of people in the low status group smoked than in the high status group . Non-smoking adolescents with a low SES started smoking more often as young adults, and young smokers with a low SES quit smoking slightly less frequently than those with a high SES. However, these social differences of individual changes are not statistically significant.The results demonstrate that smoking behaviour remains relatively stable during the transition from adolescence to young adulthood; this finding is in line with the existing literature . A largeTrend analyses of the cross-sectional data from KiGGS show that smoking prevalence rates among adolescents in Germany have steadily decreased over the past ten years . However


Salmo salar m. sebago). Relative to standard (plain) rearing tanks, provision of enrichment improved fish condition factor and survival during the first year of rearing when most mortality was attributable to parasitic and bacterial infections. The consequent higher density in enriched tanks probably induced greater growth variation and more dorsal fin damages than found in fish of standard tanks. Possibly this was partly due to the applied changes in water level. Experimentally determined smolt migration tendency at age 3 did not differ, on average, between the rearing groups, but enriched-reared fish showed clearly less variation in total movement activity than standard-reared fish. Experimental angling in earthen ponds did not suggest divergent vulnerability between the differentially reared fish at age 3, but decreased condition during the preceding growth season increased vulnerability to fishing. Based on long-term post-stocking tag returns in large-lake fisheries, fish length at release but not rearing method affected the capture rates of fish released at age 2. When released at age 3 the fish grown in enriched environment had a higher risk to be captured with stationary gears and earlier by hook and line gears compared to standard-reared conspecifics. Earlier time of maximal smolt migration activity was associated with an increased risk of being captured. We suggest that environmental enrichment may modulate growth- and behavior-related qualities that indirectly increased the vulnerability to fishing in natural conditions but not in experimental setting. The favorable effects of enrichment on early survival encourages adopting enriched rearing practices in supportive breeding of landlocked salmon.Enrichment of rearing environment with natural elements has been suggested to improve the welfare and post-release survival of cultured fish. We studied the combined effects of shelter structures, periodical water flow and water level changes on pre- and post-release performance of critically endangered landlocked Atlantic salmon ( Enormous quantities of hatchery-reared fish are being released to natural waters to support both fisheries and conservation of fish resources, but unfortunately, often without systematic evaluation of the success of the stockings . CaptureEnvironmental enrichment, that is, addition of physical structures and complexity in the rearing tanks, has been introduced as a potential measure to improve the welfare of fish and their subsequent post-stocking survival in natural environments , 15. EnrSalmo salar m. sebago Girard) in the Finnish Vuoksi watercourse represents a critically endangered salmonid population that has relied virtually entirely on a hatchery breeding program and intensive releases of 2- and 3-year-old fish since the 1970’s due to damming of the original migration routes and redirection of water from an original major spawning river for hydropower production  and covariate fish TL . Thus, increasing fish size and immature status increased migration activity and advanced the attainment of maximal migration speed.The rearing method did not affect either of the PC score means  and a marginal negative effect for CFSept . According to the final model, ΔCF was the only significant term in explaining vulnerability to angling . Rearing treatment  and fish TL in September  were dropped from the final model as non-significant.Out of 199 fish, 33 fish (16 standard- and 17 enriched-reared) were captured at least once. CF had a complex effect on vulnerability to angling, as initial models based on size measures in March or September indicated a marginal positive effect for CFn = 255 2-yo fish). Cox regression model revealed that fish tagging length was positively associated with the risk of capture , whereas rearing method or stocking site had no effect.For the fish released at age 2, tag returns were obtained from 490 individuals. 277 tags came from standard-reared fish (14.5% return rate) and 213 from enriched-reared fish (13.9% return rate). Four fish were reported twice (released after the first capture). Hook and line fishing method was reported for 175 fish (36%), stationary gears for 286 fish (58%), and 29 tag returns (6%) had no information about the gear type used. A majority of the tags from 2-yo fish (87.6%) were returned by the end of 2012 , the aven = 271, 27.3%) than fish from the standard group . For hook and line gears, the difference to the other direction was less clear (n = 45 enriched and 55 standard fish). Most of the 3-yo fish were caught during the stocking year  and 1.061 for enriched rearing (1.013–1.112)]. In addition, enriched rearing significantly increased the risk of 3-yo fish to become captured, in relation to standard rearing .For the fish released at age 3, tag returns were obtained from 594 individuals. 266 tags came from standard-reared fish (29.8%) and 328 from (36.6%) enriched-reared fish. Most fish were captured in the southern part of the lake where water depth and clarity are greater . Hook ant485 = 6.72, p < 0.001; 3-yo: t587 = 6.26, p < 0.001) and at smaller size  with stationary gears  and at smaller size  with hook and line gears compared to standard fish , 3-yo: -0.001  and PC2 score  but not by PC1 score . Thus, earlier time of maximal activity increase was associated with an increased risk of being captured when all fishing methods were combined. This result was independent of the rearing method (non-significant interaction terms between rearing method and PCs). There was no correlation between PCs and the post-release capture time in either of the rearing groups .In total, 262 tag returns (26.6%) were obtained from the fish that were tested for migration behavior before the release. The likelihood of capture was significantly affected by rearing method  after 2 and 3 summers of rearing. As a result of better survival, realized rearing density was unintendedly higher in enriched treatment in the first year. This, together with changes in water level and the consequent changes in fish density through the rearing period, possibly contributed to the larger growth variation and higher incidence of dorsal fin damages in comparison with standard rearing. Smolt migration tendency of 3-yo fish was, on average, at the same level between the rearing groups, but the activity distribution was narrower in enriched-reared fish. Rearing method did not influence the experimentally assessed vulnerability to angling, but declining body condition during the previous summer increased the risk of becoming captured. Based on post-stocking tag returns from large lake fisheries, rearing method did not affect the capture rates when the fish were released at the age of 2 years. However, enriched-reared fish had a significantly higher risk to become captured (were caught more frequently with stationary gears) than the standard-reared fish, when the fish were released at the age of 3 years. Moreover, the enriched-reared fish were captured faster than their standard-reared conspecifics with hook and line gears, mainly trolling. Because the experimental angling only revealed a strong vulnerability effect for fish condition, our results suggest that the present rearing methods induced growth and behavior-related differences that indirectly explain differential capture rates in lake fisheries.Ichthyobodo sp. parasites and Flavobacterium sp. bacteria (first year), causing a survival difference of 21%-units between the rearing methods. Since the water to the hatchery came from a common source, survival differences between the rearing methods supposedly arose due to treatment differences. Further, the observed survival rates were consistent among the replicate tanks within treatments suggesting a marginal role for pure coincidence. Nevertheless, we cannot resolve whether the treatment differences arose from environmental factors or from differences in fish resistance and tolerance. Overall, these results are consistent with previous observations on salmonids [Flavobacterium [As expected, enriched rearing significantly increased the early survival of fish over standard rearing. Most mortality occurred during the confirmed outbreaks of almonids , 34, 46,almonids . Yet, moacterium , 49. In Larger growth variation of fish in enriched conditions in relation to standard-reared group contrasted with our presumption. This can be explained, at least in part, by the higher early survival of the enriched-reared fish because early mortality can be size-selective and thus truncate size variation . HoweverFlavobacterium infections were suspected as one (if not the main) causative agent; there were also signs of inflammation in the dorsal fin of many fish. Consequently, the higher incidence of dorsal fin erosions in enriched-reared fish could also arise, to some extent, from their better survival during the first summer, relative to standard-reared fish, reflecting traces of disease outbreaks rather than the effects of enrichment itself. High rearing density or low feed ration has been shown to incur negative effects on fin condition of salmonids [In captive rearing of salmonids, phenotypic growth variation is typically increased by social hierarchies, where dominant individuals tend to monopolize food resources at the expense of others –54. In talmonids , 22, 57.n = 495 fish) compared to the standard group . In salmonids, both smolting and maturation processes are linked to early growth rate, and a certain threshold size and adequate energy reserves must be attained in order to adopt certain developmental routes [Similarly as anadromous salmon, the studied landlocked population undergo physiological, morphological and behavioral changes related to smoltification that prepare the fish for feeding migration , 58. Durl routes , 61. Migl routes .Salmo trutta), smolt migration distance at the ages of 2 and 3 years was found to be individually highly repeatable (intraclass correlation coefficient 0.608) [It is presumable that most fish in our study had already smoltified at 2 years of age when the migration tests could not yet be arranged for logistic reasons , 63, altt 0.608) . AlthougThe post-stocking tag-recovery data revealed that the proportion of tag returns was substantially higher for the fish stocked as 3-yo (approx. 30%) than for fish stocked as 2-yo (14%). However, most of the 3-yo stocked fish were caught within six months of the release, and the relative number of fish captured at smaller than the minimum size limit (500 mm) was also much larger for 3-yo (61%) than for 2-yo group (35%). Irrespective of the release age, the captures of undersized fish were mostly attributable to stationary gears (gillnets). The average time between the release and capture was also shorter in fishing with stationary gears than in hook and line fishing, mainly trolling. Yet, in reality the number of undersized fish caught using hook and line gears was probably much larger than what the tag returns indicated, because in most cases such fish can be released quickly back to the water without noting the tag or registering its code.The number of tag returns from 3-yo fish captured with stationary gears was clearly higher for the enriched (271) than for the standard group (195). Moreover, there was a gear category -dependent difference in capture rates of 3-yo enriched-reared fish, relative to the standard-reared group . This was explained by the significantly earlier average capture time of fish in the enriched rearing group by hook and line gears. These differences may result from 1) different behavior (foraging and moving activity), 2) different survival or 3) different spatial distribution of the fish groups.Gadus morhua) that more variable rearing conditions  promoted exploration rate in a novel environment and recovery from a stressful experience in relation to fish reared in a plain environment. Nevertheless, behavioral responses may largely be dependent upon the enrichment type applied and the environment type after release, i.e. river vs. large lake: when Atlantic salmon juveniles received natural prey and were conditioned to threat of predation they displayed two times lower willingness for risk-taking behavior than the fish reared under standard hatchery conditions [Different behavior is a plausible explanation for capture rate differences, because high foraging activity likely increases vulnerability to hook and line gears and high moving activity vulnerability to all sorts of stationary gears –66. Rodenditions .Oncorhynchus tshawytscha) showed that a variety of environmental enrichments in raceways, including in-stream woody debris structures, overhead covers and underwater feeding, did not consistently increase smolt or smolt-to-adult survival, in comparison with conventionally reared fish [Survival differences between the groups could explain differential capture rates, particularly because the angling experiment did not suggest rearing treatment-dependent differences in the individual vulnerability of salmon to angling. There are previous indications that enriched or seminatural rearing methods may increase post-release survival in salmonids , 24, 69.red fish , 71. Betred fish , 73. Thered fish , this chIrrespective of the age at stocking, the time the fish spent in the lake before capture diminished with increasing fish length at release. This is expected when fishing habits or regulations imply positively size-selective fishing, i.e. the faster the fish grow the faster they recruit to fishing. Hyvärinen and Vehanen  found a Sander lucioperca) [The results of this study have significant implications for the management of landlocked salmon fisheries. The undersized (3-yo) fish were vulnerable to a strong fishing pressure by size-selective and lethal gillnets, presumably increasing instant or nearly instant post-release mortality. The prevalent use of gillnets with small mesh sizes in Lake Höytiäinen is mainly due to unrestrained recreational and commercial fishing for pikeperch (ioperca) . FinallyS1 TableMeans and variations in total length, body weight and condition factor, and the percentage of matured males in standard and enriched rearing groups of 3-year-old salmon.(DOCX)Click here for additional data file.S2 TableNumber of enriched- and standard-reared 3-year-old salmon in different fin erosion categories.(DOCX)Click here for additional data file.S1 FigVariation of water temperature during the study period at Kainuu Fisheries Research Station.(DOCX)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.


In several biomedical applications, the detection of biomarkers demands high sensitivity, selectivity and easy-to-use devices. Organic electrochemical transistors (OECTs) represent a promising class of devices combining a minimal invasiveness and good signal transduction. However, OECTs lack of intrinsic selectivity that should be implemented by specific approaches to make them well suitable for biomedical applications. Here, we report on a biosensor in which selectivity and a high sensitivity are achieved by interfacing, in an OECT architecture, a novel gate electrode based on aptamers, Au nanoparticles and graphene hierarchically organized to optimize the final response. The fabricated biosensor performs state of the art limit of detection monitoring biomolecules, such as thrombin-with a limit of detection in the picomolar range (≤ 5 pM) and a very good selectivity even in presence of supraphysiological concentrations of Bovine Serum Albumin (BSA-1mM). These accomplishments are the final result of the gate hierarchic structure that reduces sterich indrance that could contrast the recognition events and minimizes false positive, because of the low affinity of graphene towards the physiological environment. Since our approach can be easily applied to a large variety of different biomarkers, we envisage a relevant potential for a large series of different biomedical applications. This is because bioelectronic devices based on biocompatible organic conductors are endowed with a mixed ionic-electronic conduction that makes them suited for the biointerfacing and allows the design of electronic devices able to implement an efficient ion-to-electron transduction, useful for their interfacing with the standard electronics. The latter property is pivotal for a special class of transistors, i.e. the Organic Electrochemical Transistors (OECTs), which are ideal candidates for several applications in bioelectronics2. OECTs’ operation relies on a change of the device output current upon the interaction of their active channel with charged species dispersed in an electrolyte, in direct contact with it. Such interaction is mediated by the action of a gate electrode immersed in the same electrolyte. OECTs are currently experiencing a renewed interest in bioelectronics. Indeed, we are witnessing a development of novel strategies, straightforward and powerful at the same time, conferring the needed selectivity for the recognition of ionic species dispersed, at low concentrations, in complex biological fluids4 . This is because OECTs lack of intrinsic selectivity: the benchmark organic transducer they are based on, i.e. the poly:polystyrene sulfonate (PEDOT:PSS), accomplishes an efficient transduction of all charged species in the electrolytic medium due to a de-doping effect occurring upon their gate voltage-driven diffusion into its bulk5. Hence, several strategies towards selectivity have been proposed in literature and particularly developed in the last years. OECT specificity has been achieved basically by pursuing two approaches, the first one consisting of device interfaces functionalization7, while the second one involves novel measurement protocols9. The first strategy, representing the most popular approach in literature, has led to a large variety of devices upon functionalization of the gate electrode or of the device channel10. The development of OECT-based immunosensors, implemented via gate functionalization, for label-free transduction of bio-recognition events is really promising for promoting a combined improvement of selectivity/sensitivity12. The second approach, indeed, even though not very common, seems to be appropriate for discriminating different analytes in biological fluids. It is based, for example, on the use of AC measurements where dopamine detection can be implemented by applying a gate voltage pulse and collecting the OECT current/transconductance responses, which show phase angle shifts that are dependent on the analyte concentration in the electrolyte9, or on the appropriate choice of transconductance, a parameter expressing the amplification capability by transistors, as figure of merit for the selective detection of different analytes mixed in a physiological environment. In this case, the transconductance as a function of the gate voltage shows multiple peaks whose positions have been associated to each specific analyte, specifically dopamine, ascorbic acid and uric acid, dissolved in an electrolytic medium8. The demanding requirement for even better performing OECTs biosensors in terms of selectivity and Limit of Detection (LoD) can be addressed by further implementing novel and promising bioreceptors. There is an increasing interest in aptamers, massively used in impedimetric aptasensors13. Aptamers are synthetic nucleic acid sequences (short single-stranded DNA or RNA) endowed with specific bio-recognition abilities through their tight binding with non-nucleic acid targets. Their strong affinity with specific target molecules is promoted by different secondary structures, such as stems, loops or tertiary motifs as G-quadruplexes, that favor binding with target analytes through different physico-chemical interactions, including van der Waals interactions, hydrogen bonding and 15. In this way, aptasensors may accomplish the detection of analytes up to the femtomolar16 and attomolar level17. The aptameric approach in the context of OECT biosensing is up to now limited to few recent papers. Chen et al. have explored the detection of glycan expression, i.e. an integral membrane protein ruling cell-to-cell interaction, on living cancer cells down to 10 cells/μL18. They have exploited a complex gate electrode interface and, analyzing the OECT time response upon subsequent injection of analyte aliquots in the gate electrolytic medium, observed an effective gate voltage shift upon recognition events. Saraf et al., based on the same type of data analysis of an effective gate voltage shift19, were able to detect epinephrine down to the picomolar level by exploiting epinephrine binding aptamers immobilized on Ti/Au gate electrodes20. Liang et al. have demonstrated the possibility of selectively detect adenosine triphosphate (ATP) with ultrahigh sensitivity down to the concentration of 10 pM21. The real time approach is expected to be time effective and, consequently, able to enhance the sensor swiftness. However, it realizes the well-known competition between swiftness and sensitivity in sensors, the latter being the minimum variation of analyte concentrations able to produce a variation of the sensing parameter. In fact, this protocol provides a short incubation time, implying a less effective coordination and recognition of the analyzed biomolecule. In addition, the memory of the past binding events during the subsequent additions of analyte aliquots in the electrolyte is expected to reduce the electrode surface still available for biorecognition events, thus reducing the sensor sensitivity. Here we report a detailed study aimed at a very sensitive and selective detection of thrombin, a protein involved in the blood coagulation, down to the picomolar level. Our experiments have been implemented on a OECT aptasensor based on PEDOT:PSS channels and specific gold nanoparticles/polyethylene multi-layers graphene (AuNPs-PMLG) gate electrodes22 functionalized by immobilizing the Thrombin Binding Aptamer . This aptamer, carrying the sequence 5′GGTTGGTGTGGTTGG3′, adopts a stable chair-like, antiparallel G-quadruplex (G4) structure and strongly inhibits fibrin clot formation by tightly binding to thrombin23. It has attracted huge attention as effective thrombin activity modulator, representing one of the most extensively studied systems in the development of thrombin-targeting assays24, and it has been also incorporated in nanosystems and nanoparticles27. The performance of our sensing devices has been carefully investigated by means of several techniques aimed at testing the fabrication process quality for the AuNPs-PMLG/TBA gate electrode. We discuss the effectiveness of such electrode in determining a hierarchic geometry, which is expected to synergistically combine a large surface area for the direct immobilization of aptameric layer with a graphene-based multilayer. The latter is characterized by the highest mechanical strength, an excellent electrical conductivity, an high surface area, a good biocompatibility and, above all, a low affinity towards undesired molecules that can aspecifically adsorb on metallic electrodes, allowing to reduce false positive results and to reach an even marked selectivity upon detecting very low levels of thrombin in presence of supraphysiological amounts of Bovine Serum Albumin (BSA), i.e. at a concentration of 1mM, being BSA the most abundant serum protein with a physiologic concentration of about 600 μM29. Our approach, taking advantage of an AuNPs-PMLG composite, is also reinforced by the choice of a novel sensing parameter, the 30. The use of the 31.Medical science is currently taking advantage from a wide set of organic electronic devices implementing, for instance, medical diagnostics/careWe fabricated an OECT sensor integrating functionalized polyethylene multi-layer graphene (PMLG) gate electrodes in a standard organic electrochemical transistor structure Fig. A. ConsisThe transistor response when gated with the bare PMLG electrode has been characterized using PBS  as electrolyte.32, at low voltage operating conditions, which is an important requirement for electronic device applications in the biological field. Excluding the very low voltage , corresponding to a reduced potential drop at the electrode/electrolyte interface, but also a more  or less  effective polarizable character towards the gate electrolyte, causing a significant potential (and gate current) drop at the gate electrode/electrolyte interface. From our characterization it is worth to note that the strong contribution of Corresponding transfer curves, obtained monitoring the evolution of mentclass2pt{minimmentclass2pt{minim35. AuNPs, prepared using this seed-induced growth method  is an effective method to enhance the conductivity and the sensing performance of electrodes. In addition, NPs are often used as anchoring elements for different compounds, being able to efficiently link chemical groups by generating strong bonds. However, it is extremely important to have NPs with a homogenous dimensional distribution to ensure an efficient decoration and controllable properties of the final electrode. We adopted the Frens protocol for the realization of AuNPs with finely tunable size, shape and electrical properties36, reveals an electron transfer process between AuNPs and the graphene electrode37 and is demonstrated by the appearance of a well-defined peak at 0.1 V in the gate current profile  studies were performed on three samples: the bare PMLG electrode, the AuNPs-PMLG electrode and the AuNPs-PMLG decorated with the TBA15 46, C–N in the nucleobases and C-O in the sugars of the oligonucleotide backbone (at 286.1 eV)47. Moreover, further features are present at 287.00 eV, related to N–C=O bond in the two basis48 and at 287.79 eV that can be attributed to the C–NH49. An additional peak at 288.71 eV is likely arising from a shakeup electron promotion process, typical of large organic molecules50. A more detailed analysis of XPS spectra  and graphene54. The aptamer immobilization on the decorated electrode impedes the electron exchange between AuNPs and the electrode52, promoting the transfer process borne by the Fc label. This fact represents an indirect evidence of the aptameric functionalization. However, the low peak intensity suggests that the Fc label transfer cannot compensate the intrinsic screening effect due to the aptameric functionalization, whose major effect is to markedly increase the electron transfer resistance of the electrode56. This fact leads to a lower 11.The transfer and the related gate current curves for this functionalized electrode, acquired prior to carry out the Thr recognition, are reported in Fig. 57. This also indicated that the sought optimal concentration was 1 μM.The weaker followed  section.entclass1pt{minima58. The resulting 52. This marked variation in the transfer curves in terms of both shape and modulation can be considered as the direct effect of the thrombin detection. To evaluate the linear range and the detection limit of the OECT based biosensor against thrombin, the calibration curve upon testing different thrombin concentrations (from 1 to 100 pM), was determined. The device response, reported in Fig. 11, since it excludes from the analysis all possible effects deriving from the aptamer, AuNPs and gate substrate and it only allows the evaluation of the thrombin coordination to the OECT sensor response. The 29. The aptasensor selectivity has been evaluated in worst conditions, i.e. at a supraphysiological BSA concentration of 1 mM. The 30. Their results demonstrated a variation in the response of about 1/2 (Rct(Thr) 59. The introduction of AuNPs with their large specific surface area on a metallic disk electrode60provides better selectivity, obtaining a ratio of 1/8 between BSA and Thr signals, but in this case the ratio between tested concentrations is A control transfer curve has been recorded for the optimal aptamer concentration in order to monitor the transition between folded and unfolded TBA15-functionalized AuNPs-PMLG gate electrode Fig. B, the coto1 Fig. , were peThe optimized hierarchical organization achieved by interfacing, on graphene sheets deposited on a Polyethylene flexible film, Au nanoparticles and aptamers has been the key to develop OECTs with unprecedented selectivity as demonstrated by studies on Thrombin used as a test biomolecule. The aptasensor performance took full advantage of the properties of the developed materials and interfaces where the innovation is the gate electrode that combines a multilayer graphene sheet, almost inert towards physiological fluids, with electrocatalytically active AuNPs. The large surface area of nanoparticles has been chosen also for easily immobilizing a larger number of aptamer molecules on the surface of the electrode leaving them the ability to change conformation, making them a useful alternative for signal amplification, while the underlaying graphene sheets, being practically inert, give the advantage of minimizing false positive results. These relevant device innovations relying on the materials and interfaces here implemented were critical for the successful monitoring of thrombin, a benchmark biomolecule used as a significant case study, up to the picomolar level, moreover showing a noteworthy selectivity and absence of nonspecific interactions even when exposed to supraphysiological concentrations of BSA, an interfering protein present in blood plasma. We believe that these results further qualify OECTs as very promising devices for biomedical applications in a perspective of biocompatible, cheap and easy-to-handle systems that could contribute to the development of point of-care diagnostics. We envisage that our approach could be profitably expanded to a large number of different biomarkers, being basically limited only by the aptamers design and fabrication.61. More in detail, for the metal patterning process, AZ1518 photoresist (PR) is used in combination with a two-steps wet etch process for the selective removal of the metals. Two distinct solutions are used for the etching: A standard 2” (100) p-doped Si wafer finished with 1 μm of 62. The as-prepared gate electrode has been characterized using high-resolution scanning electron microscopy (ZEISS field emission gun scanning electron microscope) and Raman spectroscopy (Renishaw InVia Qontor)  deposited on a flexible and insulating substrate made of a low density polyethylene (LDPE) film. Briefly, the polyethylene multi-layer graphene (PMLG) films were produced through exfoliation of nanographite through thermal and ultrasonic treatments ensuring mechanical lamination of the material. The obtained powder has been suspended in an alcoholic solution and deposited on LDPE surface by means of drop castingIds at a fixed value of The OECT’s electrical characterization has been carried out using two source measure units (NI PXle-4138/9) controlled by a customized ad hoc LABVIEW code and analyzed by means of OriginLab software. Our electrical set up ensures reliable and standardized data acquisition. The typical reproducibility of OECTs is shown in Supplementary Material-Fig. 35. Briefly, a solution of tetrachloroauric acid AuNPs were synthesized following the Frens protocolThe electrophoretic deposition (EPD) method was exploited to deposit AuNPs on the PMLG gate electrode surface with two source measure units (NI PXle-4138/9) controlled by a customized LABVIEW code. Before the EPD, the PMLG gate electrode was sonicated in isopropanol for 10 min in order to clean it. The PMLG electrode was immersed into the AuNPs suspension and was used as anode, together with a chrome electrode (5 mm × 10 mm) employed as cathode. The two electrodes were placed at a distance of about 1 cm and a voltage of 40 V was applied at the anode for 15 min. Finally, the PMLG gate electrode decorated with AuNPs (AuNPs-PMLG) was thrice rinsed with Milli-Q water  was performed (electron beam acceleration voltage of 5 keV at working distance of 3.9 mm). The AuNPs-PMLG gate electrode was also characterized by Energy Dispersive X-ray spectroscopy (EDX) performed with an Oxford-Cambridge 360 Stereo Scan working at 200 KeV, in order to study the graphene nanocomposite surface coverage. The PMLG surface was investigated by scanning the electron beam over the area of interest and simultaneously acquiring the EDX spectrum, with the aim of reconstructing the spatial distribution of the C and Au signals.The 15-mer thrombin binding aptamer, used as bioreceptor with a sequence 5’ Fc-G-G-T-T-G-G-T-G-T-G-G-T-T-G-G- C6-SH 3’, was synthesized by BIOMERS (Germany) and characterized by HPLC and MALDI-TOF analyses (data given by the provider). The C6-SH at the 3’ end is a thiol group separated from the oligonucleotides sequence by a linear linker containing six carbons, while Fc at the 5’ end stands for a ferrocene residue covalently linked to the aptamer sequence and used as redox label.For the TBA immobilization onto the AuNPs-PMLG gate electrode, 10 μl of solutions with different aptamer concentrations  in sterile water were heated at 90 °C for 5 min to allow the aptamer destructuring, increaseing the thiol group accessibility. The samples were successively dipped in a bath of cold water for 15 min to freeze the aptamer conformations. Each solution was drop casted on the surface of an AuNPs-PMLG gate and dried in ambient conditions for 4 h. Finally, the prepared TBA-functionalized AuNPs-PMLG electrodes were washed twice with sterile water to remove the unbound aptamers. After the aptamer immobilization, each gate electrode was dipped in a solution of 6-mercapto-1-hexanol , with a concentration of 100 μM, for 15 min at room temperature under gentle stirring to minimize possible unspecific binding (post-blocking agent). Two washing steps with sterilized water were finally carried out.X-ray photoelectron spectroscopy (XPS) has been performed to confirm formation of the gold-thiol binding between aptamers and AuNPs on the PMLG surface. Photoelectron analyses were performed in a UHV chamber equipped with a non-monochromatized Mg KIds measured after aptamer immobilization and To evaluate the sensing performance of the OECT-based aptasensor against thrombin (Thr) protein, a calibration curve was made at different concentrations of Thr. The purified human The evaluation of the OECT-based aptasensor against thrombin was tested by drop casting 10 μl of a solution of bovine serum albumin  with a concentration of 1 mM in PBS buffer