Camiel L M De Roij Van Zuijdewijn
email: c.deroijvanzuijdewijn@amsterdamumc.nl

Paul A Rootjes1

Menso J Nubé1

Michiel L Bots3

Bernard Canaud4

Peter J Blankestijn6

Frans J Van Ittersum1

Francisco Maduell7

Marion Morena8

Sanne A E Peters9

Robinw M Andrewdavenport10

Vernooij3

Muriel P C Grooteman1

Long-term peridialytic blood pressure changes are related to mortality

Keywords: haemodiafiltration, haemodialysis, joint models, blood pressure, long-term changes, mortality

Background.

In chronic haemodialysis (HD) patients, the relationship between long-term peridialytic blood pressure (BP) changes and mortality has not been investigated.

Methods.

To evaluate whether long-term changes in peridialytic BP are related to mortality and whether treatment with HD or haemodiafiltration (HDF) differs in this respect, the combined individual participant data of three randomized controlled trials comparing HD with HDF were used time varying Cox regression and joint models were applied.

Results.

During a median follow-up of 2.94 years, 609 of 2011 patients died. As for pre-dialytic systolic BP (pre-SBP), a severe decline (≥21 mmHg) in the preceding 6 months was independently related to increased mortality [hazard ratio (HR) 1.61, P = .01] when compared with a moderate increase. Likewise, a severe decline in post-dialytic diastolic BP (DBP) was associated with increased mortality (adjusted HR 1.96, P < .0005). In contrast, joint models showed that every 5-mmHg increase in pre-SBP and post-DBP during total followup was related to reduced mortality (adjusted HR 0.97, P = .01 and 0.94, P = .03, respectively). No interaction was observed between BP changes and treatment modality.

Conclusion.

Severe declines in pre-SBP and post-DBP in the preceding 6 months were independently related to mortality. Therefore peridialytic BP values should be interpreted in the context of their changes and not solely as an absolute value.

INTRODUCTION

Worldwide, approximately 3 million patients with end-stage kidney disease (ESKD) are treated with intermittent extracorporeal dialysis techniques, including haemodialysis (HD) and haemodiafiltration (HDF) [START_REF] Canaud | Global prevalent use, trends and practices in haemodiafiltration[END_REF]. Yet, despite their lifesaving properties, both cardiovascular (CV) morbidity and mortality remain alarmingly high [START_REF] Parfrey | The clinical epidemiology of cardiac disease in chronic renal failure[END_REF]. Besides specific renal and dialysis related risk factors, such as the retention of uraemic toxins and bio-incompatibility of the extracorporeal circuit, traditional risk factors, including hypertension and diabetes, play an important role as well. In the general population, a log-linear, inverse relationship between blood pressure (BP) and CV survival has been well established [START_REF] Lewington | Age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies[END_REF]. For dialysis patients, however, this relation is less clear. Actually, in most guidelines, strict BP targets are lacking due to the absence of robust data from well-designed and sufficiently powered trials [START_REF] Shafi | Drug selection for treating hypertension in dialysis patients: more to consider than BP-lowering potency[END_REF][START_REF] Flythe | Blood pressure and volumemanagement in dialysis: conclusions froma Kidney Disease: ImprovingGlobal Outcomes (KDIGO) Controversies Conference[END_REF][START_REF] Kidney | KDOQI Clinical Practice Guideline for Hemodialysis Adequacy: 2015 update[END_REF]. Previous observational studies have shown a U-or J-shaped relationship between BP values measured just before or after dialysis (peridialytic BP) and mortality, or no relationship at all [START_REF] Zager | U" curve association of blood pressure and mortality in hemodialysis patients[END_REF][START_REF] Sarafidis | Hypertension in dialysis patients: a consensus document by the European Renal andCardiovascularMedicine (EURECA-m) working group of the European Renal Association-European Dialysis and Transplant Association (ERA-EDTA) and the Hypertension and the Kidney working group of the European Society of Hypertension (ESH)[END_REF][START_REF] Jhee | The optimal blood pressure target in different dialysis populations[END_REF][START_REF] Robinson | Blood pressure levels and mortality risk among hemodialysis patients in the Dialysis Outcomes and Practice Patterns Study[END_REF][START_REF] Foley | Blood pressure and long-term mortality in United States hemodialysis patients: USRDS Waves 3 and 4 Study[END_REF][START_REF] Bansal | Blood pressure and risk of all-cause mortality in advanced chronic kidney disease and hemodialysis: the chronic renal insufficiency cohort study[END_REF][START_REF] Inaba | Association of blood pressure with all-cause mortality and stroke in Japanese hemodialysis patients: the Japan Dialysis Outcomes and Practice Pattern Study[END_REF]. In virtually all studies, just baseline BPs or their short-term changes were evaluated. In healthy conditions, BP is controlled by cardiac output and peripheral vascular resistance, which are both influenced by various factors, including the sympathetic nervous system and the renin-angiotensin system [START_REF] Sarafidis | Hypertension in dialysis patients: a consensus document by the European Renal andCardiovascularMedicine (EURECA-m) working group of the European Renal Association-European Dialysis and Transplant Association (ERA-EDTA) and the Hypertension and the Kidney working group of the European Society of Hypertension (ESH)[END_REF]. In HD patients, however, structural and functional derangements of the CV system, including premature vascular stiffening and left ventricular hypertrophy, may severely affect the capacity of a patient to keep BP within a normal range. Furthermore, in these patients, BP is also highly volume dependent as a result of reduced urine output [START_REF] Mailloux | Hypertension in the ESRD patient: pathophysiology, therapy, outcomes, and future directions[END_REF][START_REF] Van Buren | Hypertension and hemodialysis: pathophysiology and outcomes in adult and pediatric populations[END_REF]. Due to the intermittent character of the treatment, both volume status and BP vary considerably. Generally, BP increases in the interdialytic interval due to fluid retention and declines during dialysis (intradialytic) due to obligate ultrafiltration (UF). As for the latter, both patient and dialysis-related factors may be involved, including the pathophysiological state and reactivity of the CV system, the amount of UF needed, the UF rate and the difference between the UF rate within the dialyzer and the rate of refill from the interstitial space to the circulation. from three randomized controlled trials (RCTs) comparing HD with HDF, we recently showed that both systolic BP (SBP) and diastolic BP (DBP) decrease over time [START_REF] Rootjes | Long-term peridialytic blood pressure patterns in patients treated by hemodialysis and hemodiafiltration[END_REF]. Furthermore, previously it was shown that patients treated with HDF have a superior survival when compared with HD [START_REF] Peters | Haemodiafiltration and mortality in end-stage kidney disease patients: a pooled individual participant data analysis from four randomized controlled trials[END_REF]. In the current study, we evaluated whether long-term peridialytic BP changes are related to mortality and whether the relationship between long-term BP changes and mortality differs between HD and HDF.

MATERIALS AND METHODS

Study design

The present analysis was executed with the IPD of three RCTs comparing HD with online post-dilution HDF [START_REF] Peters | Haemodiafiltration and mortality in end-stage kidney disease patients: a pooled individual participant data analysis from four randomized controlled trials[END_REF]. A fourth RCT, that also evaluated survival in HDF and HD patients, was omitted due to the absence of longitudinal peridialytic BP data [START_REF] Ok | Mortality and cardiovascular events in online haemodiafiltration (OL-HDF) compared with high-flux dialysis: results from the Turkish OL-HDF Study[END_REF]. Details of the included studies can be found elsewhere [START_REF] Maduell | High-efficiency post dilution online hemodiafiltration reduces all-cause mortality in hemodialysis patients[END_REF][START_REF] Grooteman | Effect of online hemodiafiltration on all-cause mortality and cardiovascular outcomes[END_REF][START_REF] Morena | Treatment tolerance and patientreported outcomes favor online hemodiafiltration compared to high-flux hemodialysis in the elderly[END_REF]. In short, the CONvective TRAnsport STudy (CONTRAST) included 714 ESKD patients in 27 dialysis centres in the Netherlands, Canada and Norway. The mean convection volume in HDF patients was 20.7 l/session and the median follow-up was 2.9 years. All-cause mortality was the primary endpoint. The same holds true for the Spanish Estudio de Supervivencia de Hemodiafiltración On-Line (ESHOL), which included 906 patients. In this investigation, the quarterly measured mean convection volumes in HDF patients ranged from 22.9 to 23.9 l/session and the median follow-up was 2.1 years. Lastly, the French Convective versus Hemodialysis in Elderly (FRENCHIE) study evaluated 391 patients (age ≥65 years). While intradialytic tolerance was the primary endpoint, mortality was a secondary objective. The mean reached convection volume in HDF patients ranged from 19.3 to 22.5 l/session and the median follow-up was 2.0 years. All studies randomized patients in a 1:1 ratio.

Data collection

At baseline, demographics, medical history, laboratory values and dialysis parameters were collected. Body mass index (BMI; kg/m2) was calculated as post-dialysis weight (kg)/height (m2). A history of CV disease (CVD) included myocardial infarction, angina pectoris, therapeutic coronary procedure, transient ischaemic attack, stroke, therapeutic carotid procedure (endarterectomy or stenting), vascular intervention (percutaneous transluminal angioplasty, revascularisation or stenting) and amputation.

BP measurements

At baseline and during follow-up, SBP and DBP were measured both before (pre) and after (post) dialysis by automatically inflated manometric cuffs using a digital monitor attached to the dialysis machine, according to standard protocols. Differences between the post-and pre-BP values (dialytic) were calculated by subtracting pre-BP values from post-BP values, therefore positive values represent an increase in BP during dialysis. Pulse pressure (PP) was calculated by subtracting DBP from SBP, and mean arterial pressure (MAP) by using the formula (1/3*SBP + 2/3*DBP). The average BP of three consecutive dialysis sessions was registered at baseline and every 3 (CONTRAST and ESHOL) or 6 (FRENCHIE) months thereafter. While four peridialytic BP values were measured directly [pre-dialytic SBP (pre-SBP), post-dialytic SBP (post-SBP), pre-dialytic DBP (pre-DBP) and post-dialytic DBP (post-DBP)], eight parameters were calculated: SBP, DBP, pre-PP, post-PP,PP, pre-MAP, post-MAP and MAP.

Follow-up

Several patients moved to another centre, switched to peritoneal dialysis or underwent renal transplantation, so all efforts were made to obtain complete follow-up data for all patients in this IPD analysis. Ultimately, 99.8% had complete follow-up (until death or the end of the study).

Statistical analysis

Descriptive statistics are presented as mean [standard deviation (SD)], median [interquartile range (IQR)] or number (percentage), as dictated by the data type. In all cases, model assumptions were checked and not violated. Corrected models were adjusted for age, gender, history of CVD, dialysis vintage, BMI, diabetes and, when applicable, the corresponding baseline BP (as determined by the type of BP analysed, i.e. correction for baseline pre-SBP for the analysis on the change in pre-SBP on mortality, etc.). Whenever possible, complete follow-up (i.e. intention-to-treat) was used and renal transplantation handled as a competing risk [START_REF] Hazelbag | The importance of considering competing treatment affecting prognosis in the evaluation of therapy in trials: the example of renal transplantation in hemodialysis trials[END_REF]. Analyses were performed with SPSS Statistics version 24 (IBM, Armonk, NY, USA) or RStudio version 1.1.456 (Posit Software, Boston, MA, USA). The R-package 'JM' was used to fit joint models [START_REF] Rizopoulos | an R package for the joint modelling of longitudinal and time-to-event data[END_REF]. To reduce chances of a type 1 statistical error due to multiple testing, the P-value at which a certain difference was considered statistically significant was adjusted according to the Bonferroni-Holm method [START_REF] Holm | A simple sequentially rejective multiple test procedure[END_REF].

Assessment of the relationship between BP and mortality using time-to-event models

To assess the comparability of the data with previous studies, we first evaluated the relationship between absolute baseline SBP and DBP and mortality using Cox proportional hazards models. Patients were divided into sextiles to account for a potential non-parametric relationship. The fourth sextile (lowest hazard for pre-SBP) was used as the reference category. Next, to evaluate the relationship between a 6-month BP change and mortality, Cox proportional hazards models with the 6-month BP change as a time-varying variable were used, allowing BP change to evolve over time. Patients without follow-up after 6 months were excluded from the analyses. Again, to account for a possible non-linear relationship between 6-month BP changes and mortality, we divided all 6-month BP changes in sextiles as determined by the change in the first 6 months (M6 -M0, positive values representing a BP increase). We then evaluated a number of potential interactions to determine whether the relationship between BP change and mortality differed between HD and HDF patients, HD patients and HDF patients who achieved high convection volumes (>23 l/session on average; hvHDF) [START_REF] Peters | Haemodiafiltration and mortality in end-stage kidney disease patients: a pooled individual participant data analysis from four randomized controlled trials[END_REF], patients with a high or low baseline BP and men and women. Next, Cox proportional hazards models were fitted with sextiles of 6-month BP changes as a timevarying variable and sextile 4 as the reference category. Using this approach, the relationship between the most recent (i.e. preceding) 6-month BP change and mortality could be assessed. Both crude and adjusted models were fitted. To determine whether the relationship between BP change and mortality was driven by changes in interdialytic weight gain, we fitted the adjusted models again and included the corresponding time-varying 6-month change in UF rate. Since correcting for the corresponding change in UF rate may be insufficient when dry weight increases, we plotted dry weight over time to evaluate a potential relative change in UF rate. Lastly, models were fitted as clustered by study to evaluate a potential study effect.

To increase the robustness of our findings, joint models were fitted to determine the relationship between the rate of change in BP (i.e. slope) and mortality. Details concerning joint models are described extensively elsewhere [START_REF] Rizopoulos | an R package for the joint modelling of longitudinal and time-to-event data[END_REF][START_REF] Chesnaye | An introduction to joint models-applications in nephrology[END_REF]. In short, this model combines a generalized linear mixed model (LMM) with a Cox proportional hazards model. This approach makes it possible to relate the linear long-term slope of BP to mortality. As a result, the hazard ratio (HR) per amount of BP change over time can be calculated. For the LMMs, a random intercept, slope or both were used, depending on the lowest Akaike's information criterion.

RESULTS

Baseline patient characteristics (n = 2011) are shown in Table 1. The mean age was 67.0 years and 64.0%were male. The mean pre-BP was 141/72mmHg at baseline and the mean post-BP was 132/69 mmHg. Previously, baseline characteristics stratified by the original study were determined to identify potential heterogeneity between patients in the three included trials. It appeared that patients from FRENCHIE, a study specifically designed for the elderly, were older and less frequently transplanted. Furthermore, mean pre-SBP and pre-DBP were higher in CONTRAST than in the other two studies [START_REF] Rootjes | Long-term peridialytic blood pressure patterns in patients treated by hemodialysis and hemodiafiltration[END_REF]. In total, 609 of 2011 patients died during a median followup of 2.94 years (IQR 1.93-3.00).

Potential interactions between time-varying 6-month BP change and mortality

As shown in Supplementary Table S1, all investigated interactions were not significant. Thus, the relationship between the time-varying 6-month BP change and mortality was not different between HD and HDF patients nor between patients treated with HD or hvHDF [START_REF] Peters | Haemodiafiltration and mortality in end-stage kidney disease patients: a pooled individual participant data analysis from four randomized controlled trials[END_REF] nor between patients with a high or low baseline BP nor between men and women. As a result, no stratified analyses were necessary and we continued our analyses with the pooled cohort.

Baseline BP and mortality

As can be seen from Supplementary Tables S2 andS3, the overall relationship with all BP values (pre-SBP, post-SBP, SBP, pre-DBP, post-DBP and DBP) and mortality appeared U-shaped. No significant differences between the sextiles of absolute baseline BPs and mortality were found in the adjusted models after correcting for multiple testing for pre-SBP, post-SBP, SBP, pre-DBP and post-DBP. However, in the adjusted models for DBP, both sextiles 1 and 6 had a significantly higher mortality risk when compared with sextile 4.

Time-varying 6-month BP change and mortality

SBP

Both crude and adjusted HRs (aHRs) for mortality of sextiles of the time-varying 6-month changes in SBP are visualized in Fig. 1 and shown in Supplementary Table S4. Whereas patients with a moderate decline to a severe increase in pre-SBP in the preceding 6 months had a similar mortality risk, patients with a severe decline (≥21 mmHg) had a 61% increased mortality risk {aHR 1.61 [95% confidence interval (CI) 1.13-2.28]} when compared with a moderate increase. No significant differences were observed for long-term changes in post-SBP or SBP.

DBP

In Fig. 2 and Supplementary Table S5, HRs of sextiles of the time-varying 6-month change in DBP are shown. A U-shaped relationship was observed between 6-month BP changes in pre-DBP, post-DBP and DBP and mortality. However, when compared with a stable or moderate increase, only the mortality risks in patients with severe declines in the preceding 6 months in post-DBP and DBP were statistically significant [aHR 1.96 (95% CI 1.40-2.77) and 1.69 (95% CI 1.17-2.45), respectively).

MAP and PP

In Supplementary Tables S6 andS7, the results of both crude and adjusted models are shown for the relations between the 6-month changes in MAP and PP and mortality. Only a severe declining post-MAP was related to increased mortality when compared with stable or mildly increasing values [aHR 1.71 (95% CI 1.18-2.46)]. No specific shape in the relationship between a changing pre-PP or post-PP and mortality could be identified. In contrast, a severe increase in PP in the preceding 6 months was related to increased mortality when compared with stable or mildly increasing values [aHR 1.89 (95% CI 1.30-2.74)].

Additional analyses

All Cox regression analyses adjusted for the 6-month change in UF rate yielded similar results. Significant longitudinal changes in dry weight were not observed. Lastly, analyses clustered by study also yielded similar results (data not shown).

Joint models

In Table 2, the HRs per 5 mmHg increase in BP during follow-up are summarized.

SBP

For pre-SBP, the aHR for mortality was 0.97 (95% CI 0.94-0.99) for every 5-mmHg increase. Furthermore, a similar increase in post-SBP was related to improved survival [aHR 0.95 (95% CI 0.92-0.98)]. No relationship was found between long-term changes in SBP and mortality.

DBP

Every 5 mmHg increase in post-DBP was related to a 6% decrease in mortality risk [aHR 0.94 (95% CI 0.88-1.00)]. Although an increase in pre-DBP was associated with a reduced mortality risk in a crude model, the difference did not reach statistical significance in the adjusted model. Interestingly, an increase in DBP was also related to improved survival [aHR per 5 mmHg increase 0.88 (95% CI 0.80-0.97)].

MAP and PP

In the adjusted models, both increases in post-MAP [aHR per 5 mmHg increase 0.93 (95% CI 0.88-0.98)] and pre-PP [aHR per 5 mmHg increase 0.96 (95% CI 0.93-0.99)] were related to a reduced mortality risk.

DISCUSSION

In the present study, we analysed whether changes in peridialytic BP values in the preceding 6 months are independently related to mortality. Furthermore, multiple potential interactions, including differences in the relationship between longitudinal BP changes and mortality for patients treated with different dialysis modalities (i.e. HD versus HDF) were evaluated. As for the primary objective, it clearly appeared that severe declines in both pre-SBP and post-DBP in the preceding 6 months are related to an increased mortality risk, irrespective of age, underlying comorbidity, UF volume changes, dialysis vintage and BMI. The relationship between a declining peridialytic MAP and mortality is a reasonable consequence of these findings. In contrast, every 5 mmHg increase during follow-up in both pre-SBP and post-DBP was related to a 3% and 6% survival advantage, respectively. Decreasing BP in a population with a high prevalence of fluid overload could result from a loss of total body water due to forced UF [START_REF] Zoccali | Chronic fluid overload and mortality in ESRD[END_REF]. Yet the finding that decreasing BP is associated with mortality, even in patients whose UF volume remained stable or increased, suggests that fluid underfill does not underlie these results. Second, our analyses showed that the relationship between BP changes in thepreceding6months and mortality was similar for patients treated with HD or HDF and for patients treated with HD or hvHDF.

An important clinical consequence of our findings is that peridialytic BP values should not be interpreted in absolute values, but in the context of their longitudinal changes. In fact, most previous studies have focused on the relationship between an absolute BP value and mortality in search of specific BP targets. Results ranged from J-shaped [START_REF] Inaba | Association of blood pressure with all-cause mortality and stroke in Japanese hemodialysis patients: the Japan Dialysis Outcomes and Practice Pattern Study[END_REF] to U-shaped relations [START_REF] Zager | U" curve association of blood pressure and mortality in hemodialysis patients[END_REF][START_REF] Jhee | The optimal blood pressure target in different dialysis populations[END_REF][START_REF] Robinson | Blood pressure levels and mortality risk among hemodialysis patients in the Dialysis Outcomes and Practice Patterns Study[END_REF][START_REF] Bansal | Blood pressure and risk of all-cause mortality in advanced chronic kidney disease and hemodialysis: the chronic renal insufficiency cohort study[END_REF], or no relationship at all [START_REF] Foley | Blood pressure and long-term mortality in United States hemodialysis patients: USRDS Waves 3 and 4 Study[END_REF]. Yet, virtually all studies are not only limited by their observational design, but also by the assessment of a single baseline BP measurement or the mean baseline BP of a few sessions or a short term change, limited power and/or a limited follow-up period [START_REF] Inrig | Association of blood pressure increases during hemodialysis with 2-year mortality in incident hemodialysis patients: a secondary analysis of the Dialysis Morbidity and Mortality Wave 2 Study[END_REF][START_REF] Yang | Post dialysis blood pressure rise predicts longterm outcomes in chronic hemodialysis patients: a four-year prospective observational cohort study[END_REF][START_REF] Zhangh | Association of all-cause mortality with pre-dialysis systolic blood pressure and its peridialytic change in chronic hemodialysis patients[END_REF][START_REF] Inrig | Decreased pulse pressure during hemodialysis is associated with improved 6-month outcomes[END_REF][START_REF] Sipahioglu | Early systolic blood pressure changes in incident hemodialysis patients are associated with mortality in the first year[END_REF][START_REF] Raimann | Blood pressure stability in hemodialysis patients confers a survival advantage: results from a large retrospective cohort study[END_REF]. To assess whether our patient cohort is comparable to those in prior reports, we also evaluated potential associations between baseline BP parameters and mortality. The results of this exercise were highly consistent with the abovementioned studies (Supplementary Tables S2 andS3). Yet, as mentioned, our primary objective was to assess whether long-term changes in peridialytic BP values were associated with mortality. Only one study evaluated the association between PP, measured as a quarterly time-varying variable, and mortality, and found a U-shaped relationship [START_REF] Lertdumrongluk | Changes in pulse pressure during hemodialysis treatment and survival in maintenance dialysis patients[END_REF], suggesting that a stable PP is associated with superior survival. To the best of our knowledge, no previous study has evaluated the long-term changes in peridialytic SBP, DBP or its derivatives (MAP, PP) in relation to mortality using the current sophisticated approach.

The second objective of our study was to assess whether a relatively stable peridialytic BP profile may contribute to the superior survival of HDF versus HD [START_REF] Peters | Haemodiafiltration and mortality in end-stage kidney disease patients: a pooled individual participant data analysis from four randomized controlled trials[END_REF][START_REF] Nube | Mortality reduction by post dilution onlinehaemodiafiltration: a cause-specific analysis[END_REF]. Apart from the removal of larger uraemic solutes [START_REF] Penne | Role of residual kidney function and convective volume on change in beta2-microglobulin levels in hemodiafiltration patients[END_REF] and less inflammation [START_REF] Den Hoedt | Online hemodiafiltration reduces systemic inflammation compared to low-flux hemodialysis[END_REF], it has been suggested that haemodynamic factors might play a role in this respect. Previously we showed that peridialytic BP changes are similar in both modalities [START_REF] Rootjes | Long-term peridialytic blood pressure patterns in patients treated by hemodialysis and hemodiafiltration[END_REF]. Those findings, combined with the present finding that the relationship between BP changes and mortality is similar for HD and both HDF modalities, make it unlikely that the advantageous effect of HDF on survival is due to a superior long-term peridialytic BP profile.

From the joint models in the present study, it appears that an increase in pre-SBP and/or post-DBP over time is related to a lower mortality rate, independent of comorbidity or changes in UF volume and BMI. Yet, whether these findings represent the natural slope in (surviving) ESKD patients or result from interventions in fluid management and/or medication is a matter of speculation, since reliable information on these items was absent. Since dialysis patients take two to three antihypertensive drugs on average [START_REF] Morais | Fluid overload is associated with use of a higher number of antihypertensive drugs in hemodialysis patients[END_REF], and we were unable to analyse their longitudinal changes, it is premature to formulate long-term BP change targets based on the present analysis. Nonetheless, as improvements in CVD are unlikely in this population and the relationship between BP changes and survival was independent of UF volume, increasing BP may be explained by a reduction in antihypertensive medication and/or changes in the diet (e.g. an increase in sodium intake). Since ESKD patients often suffer from compromised microcirculation [START_REF] Thang | Premature aging of the microcirculation in patients with advanced chronic kidney disease[END_REF] and intradialytic hypotensive periods are common [START_REF] Flythe | Association of mortality risk with various definitions of intradialytic hypotension[END_REF][START_REF] Rootjes | Highvolume hemodiafiltration and cool hemodialysis have a beneficial effect on intradialytic hemodynamics: a randomized cross-over trial in four intermittent dialysis strategies[END_REF], it is conceivable that a modest BP increase has a dampening effect on ischaemic injury during treatment. Thus, if anything, a reduction in antihypertensive medication may be salutary for patients with long-term decreasing peridialytic BP. Most likely, the association between a long-term peridialytic BP decrease and mortality is caused by a further deterioration of pre-existing CV derangements and/or the cumulative effect of repetitive intradialytic hypoperfusion of vital organs [START_REF] Inrig | Association of intradialytic blood pressure changes with hospitalization and mortality rates in prevalent ESRD patients[END_REF]. Future studies could evaluate the possibility to predict mortality with peridialytic BP changes.

The present study has several limitations and strengths. First, the present analysis should be considered an observational study, thus residual confounding cannot be excluded. Second, from a methodological point of view, it is important to emphasize the limitations of the relatively new joint models, which combine LMM with time-to-event analyses. An LMM generates a linear slope (i.e. a fixed difference over a fixed period of time), which is then related to mortality. As our time-varying Cox regression models indeed showed a nonlinear relationship between BP changes and mortality, the currently estimated effects of the joint models may in fact be an underestimation of the true effects at the upper and lower ends of the changes. After all, if the true hazard of a decrease in BP is actually exponential above a certain threshold, the generated linear slope has a dampening effect on the estimated HR when compared with the true hazard. Furthermore, only peridialytic BP changes were analysed. Previous research indicated that interdialytic BP measurements may have more prognostic impact than peridialytic assessments at the dialysis unit. However, measuring interdialytic BP values requires expensive equipment that is not available for all patients. Furthermore, BP self-assessment may be challenging for elderly or frail patients. In contrast, BP measurements by the dialysis machine two to three times per week are an attractive and readily available alternative. Moreover, currently it is unclear whether changes in peridialytic BP values are just as predictive for survival as absolute interdialytic BP measurements. Finally, the lack of information on fluid status and on antihypertensive drugs is an important limitation for the appraisal of long-term BP declines. Yet correction for changes in BMI or UF volume did not alter the outcome, meaning that BP declines were independent of their bidirectional changes. Important strengths of this study are the large number of patients, the meticulous prospective data collection and the long and complete follow-up until death or the end of the study for 99.8% of all patients. The various statistical approaches increase the robustness of our findings. Lastly, as the study included patients from 88 dialysis facilities in five countries, our results appear generalizable to a large proportion of the dialysis population.

In conclusion, severe declines in pre-SBP and post-DBP in the preceding 6 months are related to increased mortality, independent of dialysis modality, UF rate and BMI. Therefore, peridialytic BP values should be interpreted in the context of their long-term changes and not just on an absolute value or on short-term monitoring.

ACKNOWLEDGEMENTS

The following persons participated in the HDF Pooling Project: Michiel L. Bots (Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands); Peter J. Blankestijn (Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands); Bernard Canaud(Center of Excellence Medical, Fresenius Medical Care GmbH, Bad Homburg, Germany and University of Montpellier, Research and Training Unit Medicine, Montpellier, France); Andrew Davenport (Royal Free Hospital, University College London Medical School, London, UK); Muriel P.C. Grooteman and Menso J. Nubé (Amsterdam UMC, location Vrije Universite Amsterdam, Nephrology, Amsterdam, The Netherlands and Amsterdam Cardiovascular Sciences, Diabetes & Metabolism, Amsterdam, The Netherlands); Sanne A.E. Peters (George Institute for Global Health, University of Oxford, Oxford, UK); Marion Morena (PhyMedExp, INSERM, CNRS, University of Montpellier, Département de Biochimie et Hormonologie, CHU Montpellier, Montpellier, France); Francisco Maduell and Ferran Torres (Department of Nephrology, Hospital Clinic, Barcelona, Spain); Ercan Ok and Gulay Asci (Division of Nephrology, Ege University School of Medicine, Izmir, Turkey) and Francesco Locatelli (Department of Nephrology, Alessandro Manzoni Hospital, Lecco, Italy).

FUNDING S.A.E.P. and the meetings of the representatives of the combined authors of CONTRAST, ESHOL and FRENCHIE were financially supported by the EuDial working group. EuDial is an official working group of the European Renal Association-European Dialysis Transplant Association (https://www.era-online.org/about-us/working-groups/ eudial-working-group/). No industry funding was received for any part of or activity related to the present analysis.

DATA AVAILABILITY STATEMENT

The data underlying this article will be shared upon reasonable request to the corresponding author.

AUTHORS' CONTRIBUTIONS

C.L.M.d.R.v.Z. and P.A.R. were responsible for writing the manuscript and statistical analysis. M.P.C.G., M.J.N., F.J.V.I., A.D. and B.C. were responsible for critical review of the manuscript. M.L.B., S.A.E.P., R.W.M.V., M.M., F.M. and P.J.B. were responsible for data acquisition/provision. M.J.N. and M.P.C.G. were responsible for the research idea and study design. Each author contributed important intellectual content during manuscript drafting or revision and agrees to be personally accountable for the individual's own contributions and to ensure that questions pertaining to the accuracy or integrity of any portion of the work, even one in which the author was not directly involved, are appropriately investigated and resolved, including with documentation in the literature if appropriate.

CONFLICT OF INTEREST STATEMENT

P.A.R. reports grant support from Niercentrumaan de Amstel, Elyse Klinieken and B. Braun Avitum. B.C. is part-time employee of Fresenius Medical Care acting as a consultant. F.J.V.I. reports a fee from Alfa Sigma. F.M. reports consulting fees from Fresenius Medical Care and Baxter as well as honoraria from Medtronic and Nipro. S.A.E.P. reports support from a UK Medical Research Council Skills Development Fellowship. M.P.C.G. reports grant support from Niercentrumaan de Amstel, Elyse Klinieken and B. Braun Avitum; speaker's fees from Fresenius Medical Care (payment made to the institution) and royalty fees from Wolters Kluwer. M.J.N. reports grant support from Niercentrumaan de Amstel, Elyse Klinieken and B. Braun Avitum. The remaining authors report no conflicts of interest. The authors declare that the results presented in this article have not been published previously in whole or part, except in abstract format.


Joseph Boulos

Firat Goc

Noémie Perret

Franck Rataboul

Eric Marceau

Jeremy Dhainaut

Sebastien Royer

Cellulose conversion to glycols over DUT-8

come

Cellulose conversion to glycols over DUT-8(Ni) derived nickeltungsten/carbons: selectivity tuning.

Joseph Boulos, [START_REF] Zhang | A new 3D mesoporous carbon replicated from commercial silica as a catalyst support for direct conversion of cellulose into ethylene glycol[END_REF] Firat Goc, [START_REF] Yang | Versatile nickel-tungsten bimetallics/carbon nanofiber catalysts for direct conversion of cellulose to ethylene glycol[END_REF] Noémie Perret, [START_REF] Yang | Versatile nickel-tungsten bimetallics/carbon nanofiber catalysts for direct conversion of cellulose to ethylene glycol[END_REF] Franck Rataboul, [START_REF] Yang | Versatile nickel-tungsten bimetallics/carbon nanofiber catalysts for direct conversion of cellulose to ethylene glycol[END_REF] Éric Marceau, [START_REF] Zhang | A new 3D mesoporous carbon replicated from commercial silica as a catalyst support for direct conversion of cellulose into ethylene glycol[END_REF] Jérémy Dhainaut, [START_REF] Zhang | A new 3D mesoporous carbon replicated from commercial silica as a catalyst support for direct conversion of cellulose into ethylene glycol[END_REF] Sébastien Royer .gW -1 was reported, which is two orders of magnitude higher than previously reported Ni-W-based catalysts [2]. Interestingly Sun et al. [3] showed the impact of the Sn phase and valence on the catalytic properties of bimetallic systems supported on activated carbon. When powder of metallic Sn was used with Ni/AC, a Ni-Sn alloy formed which promoted retro-aldol cleavage and hydrogenation, finally favoring EG production (58 %). When SnO was used, the catalyst promoted glucose isomerization, leading preferentially to PG (32 %). The above studies inspired our current work. A series of W and Nicontaining metal-organic frameworks were constructed by one-pot assembly of DUT-8(Ni) MOF precursors and Na2WO4•2H2O. A subsequent pyrolysis of the W@ DUT-8(Ni) materials at 700 °C under nitrogen produced nickel-tungsten/carbon catalysts. The following nomenclature was adopted for the resulting materials: NiW-l-x-C-N2; where x (nW/nNi) was set at 0.06, 0.12, 0.3, and 0.43, respectively. Figure 1 displays the catalytic results of the as-synthesized materials in the cellulose hydrogenolysis at 245 °C in a reaction time of 1 hour. Both materials with low (NiW-l-0.06-C-N2) and high (NiW-l-0.3-C-N2) W content were selective to EG (17 %). A remarkable yield switch to PG (36 %) was obtained at an average W loading (NiW-l-0.12-C-N2). The highest EG molar yield (25 %) was obtained over NiW-l-0.43-C-N2.

Figure 1 .

 1 Figure 1. Products' molar yields over NiW catalysts having different tungsten loadings. Reaction conditions: 0.50 g cellulose, 0.15 g catalyst, 50 mL water, 60 bar H2, 1000 rpm, 245°C, and 1 hour.


Danielly S Rocha

Richard P S De Campos

Habdias A Silva-Neto

Gerson F Duarte-Junior

Fethi Bedioui

Professor Wendell K T Coltro
email: wendell@ufg.br

Digital microfluidic platform assembled into a home-made studio for sample preparation and colorimetric sensing of S-nitrosocysteine

Keywords: luminosity control, droplet movement, on-chip decomposition, colorimetric sensor, Snitrosothiols

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

In recent years, many technological efforts have been paid to simplify and automate the liquidhandling process aiming integrating multiple analytical steps on single chip-based platforms to achieve high-throughput microfluidic systems with sample-in-answer-out capability [START_REF] Battat | Nonlinear Phenomena in Microfluidics[END_REF][START_REF] Samiei | A review of digital microfluidics as portable platforms for lab-on a-chip applications[END_REF][START_REF] Malic | Integration and detection of biochemical assays in digital microfluidic LOC devices[END_REF][START_REF] Haeberle | Microfluidic platforms for lab-on-a-chip applications[END_REF][START_REF] Fair | Digital microfluidics: Is a true lab-on-a-chip possible?[END_REF].

However, conventional microfluidic devices often rely on external equipment such as micro-pumps or high voltage power supplies to promote precise handling of fluids on microscale, which can be challenging depending on the demand of experiments [START_REF] Battat | Nonlinear Phenomena in Microfluidics[END_REF][START_REF] Haeberle | Microfluidic platforms for lab-on-a-chip applications[END_REF][START_REF] Whitesides | The origins and the future of microfluidics[END_REF][START_REF] Battat | An outlook on microfluidics: The promise and the challenge[END_REF][START_REF] Noviana | Microfluidic Paper-Based Analytical Devices: From Design to Applications[END_REF][START_REF] Gervais | Microfluidic chips for point-of-care immunodiagnostics[END_REF].

In this scenario, digital microfluidics (DMF) have emerged as powerful platforms for promoting the automated and programable liquid handling in well-defined droplets based on application of controlled potential to an array of actuating electrodes [START_REF] Samiei | A review of digital microfluidics as portable platforms for lab-on a-chip applications[END_REF][START_REF] Choi | Digital microfluidics[END_REF][START_REF] Narahari | Portable sample processing for molecular assays: application to Zika virus diagnostics[END_REF][START_REF] Sklavounos | Digital Microfluidic Hemagglutination Assays for Blood Typing, Donor Compatibility Testing, and Hematocrit Analysis[END_REF]. Due to its versatility, DMF technology offers reconfigurability of experiments and automation integrated on a same platform [START_REF] Choi | Digital microfluidics[END_REF][START_REF] Zhang | Advances in integrated digital microfluidic platforms for point-of-care diagnosis: a review[END_REF], allowing performing multiple analytical procedures in a sequential mode including droplet sampling, mixing, splitting, and merging, thus simplifying the day-to-day laboratory routine [START_REF] Jebrail | Digital microfluidics: A versatile tool for applications in chemistry, biology and medicine[END_REF][START_REF] De Campos | plug-n-Play" Sensing with Digital Microfluidics[END_REF], and becoming highly attractive for applications in bioanalysis and point-of-care testing [START_REF] Zhang | Advances in integrated digital microfluidic platforms for point-of-care diagnosis: a review[END_REF][START_REF] Li | Point-of-care blood coagulation assay enabled by printed circuit board-based digital microfluidics[END_REF][START_REF] Antkowiak | Integrating DNA Encapsulates and Digital Microfluidics for Automated Data Storage in DNA[END_REF].

As highlighted in the literature, DMF platforms can be embedded with different detection methods [START_REF] Malic | Integration and detection of biochemical assays in digital microfluidic LOC devices[END_REF], providing potentiality for multi-assay approaches and reproducible routine analysis, thereby reducing operational user mistakes. Additionally, some inherent advantages of DMF are the use of low quantities of samples and reagents, portability, and design versatility. The DMF technology is compatible for integration with a variety of detection methods such as mass spectrometry [START_REF] Kirby | Digital microfluidics: An emerging sample preparation platform for mass spectrometry[END_REF], nuclear magnetic resonance [START_REF] Swyer | Interfacing digital microfluidics with high-field nuclear magnetic resonance spectroscopy[END_REF], electrochemical detection [START_REF] De Campos | plug-n-Play" Sensing with Digital Microfluidics[END_REF], spectroscopic techniques [START_REF] Kalsi | A programmable digital microfluidic assay for the simultaneous detection of multiple anti-microbial resistance genes[END_REF][START_REF] Li | Intelligent droplet tracking with correlation filters for digital microfluidics[END_REF][START_REF] Gu | Integrated Digital Microfluidic Platform for Colorimetric Sensing of Nitrite[END_REF] digital cameras [START_REF] Vo | Image-based feedback and analysis system for digital microfluidics[END_REF][START_REF] Komatsu | Determination of blood lithium-ion concentration using digital microfluidic whole-blood separation and preloaded paper sensors[END_REF] or smartphones [START_REF] Gu | Integrated Digital Microfluidic Platform for Colorimetric Sensing of Nitrite[END_REF][START_REF] Jafry | Double-sided electrohydrodynamic jet printing of two-dimensional electrode array in paper-based digital microfluidics[END_REF][START_REF] Yafia | Ultra-portable smartphone controlled integrated digital microfluidic system in a 3D-printed modular assembly[END_REF].

Vo et al. [START_REF] Vo | Image-based feedback and analysis system for digital microfluidics[END_REF] reported an interesting application of a DMF system combined with colorimetric detector and a white-colored box coupled with dual-branch fiber optic illuminator as an affordable analytical tool for monitoring droplet position and enzymatic colorimetric reaction. The authors showed that the assembling of camera and a zoom lens improved the colorimetric performance of enzymatic assay of β-glycosidase. In addition, it was used an image-based algorithm to analyze the enzymatic product to successfully perform the treatment and analysis of colorimetric responses. Li et al. [START_REF] Li | Intelligent droplet tracking with correlation filters for digital microfluidics[END_REF] proposed a DMF system with intelligent droplet tracking for estimating the droplet velocity on colorimetric assays using Griess reaction, as model. In the mentioned report, a correlation filter tracker was used to avoid interferences provided by background patterns or inhomogeneous illumination.

When associated to colorimetric responses, portable DMF systems are quite promising to enable the most variated chemical and biochemical analysis. In this context, promoting luminosity control and homogenous background light is essential for achieving high performance optical detection [START_REF] Li | Intelligent droplet tracking with correlation filters for digital microfluidics[END_REF].

Since some models of DMF chips are commonly based on conductive materials that present color [START_REF] Gu | Integrated Digital Microfluidic Platform for Colorimetric Sensing of Nitrite[END_REF] or high reflection [START_REF] Vo | Image-based feedback and analysis system for digital microfluidics[END_REF], the development of studies aiming to mitigate these challenges regarding colorimetric detection directly on DMF chips are extremely important in the realm of colorimetry. In addition, the development and integration of multiple analytical procedures from sample preparation to detection stages to allow a complete analysis using DMF systems can provide a powerful and portable tool for monitoring chemical species in real time [START_REF] Li | Point-of-care blood coagulation assay enabled by printed circuit board-based digital microfluidics[END_REF].

Therefore, this study proposes, for the first time, the integration of a DMF platform with a mini studio to promote a semi-automated analytical protocol towards on-chip complete analysis, i.e., from sample preparation to colorimetric detection. As proof-of-concept, the conventional colorimetric reaction involving modified Griess reagent was explored for the indirect analysis of Snitrosocysteine (CySNO), which was decomposed through exposure to UV light using blue LEDs.

For this purpose, a 3D printed support combined with UV-LED was manufactured to enable on-chip biomolecule decomposition. The portable instrumentation was used in-situ for the full sample procedures, including decomposition, reagent mixing, droplet splitting and colorimetric analysis. A mini studio was manufactured using low-cost materials and then assembled with a webcam to simplify two important processes: background lighting and pixel capture. Lastly, it is important to emphasize that CySNO is a low-molecular weight S-nitrosothiol (LMW-RSNO), carrier of nitric oxide (NO), that present important physiological contributions in the human body. In this way, the concentration of RSNOs can be determined to monitor numerous diseases, such as Parkinson's, Alzheimer's, sepsis, heart failure and asthma [START_REF] Ling | In situ synthesis of ultrafine Cu2O on layered double hydroxide for electrochemical detection of S-nitrosothiols[END_REF][START_REF] Ismail | Colorimetric analysis of the decomposition of S-nitrosothiols on paper-based microfluidic devices[END_REF][START_REF] Hess | Protein S-nitrosylation: Purview and parameters[END_REF][START_REF] Sengupta | The role of thioredoxin in the regulation of cellular processes by S-nitrosylation[END_REF].

MATERIALS AND METHODS

Chemicals and materials

Sodium nitrite, monobasic sodium phosphate, dibasic sodium phosphate, ethylenediaminetetraacetic acid (EDTA), acetonitrile, sulfanilamide, N-(1-naphthyl)ethylenediamine dihydrochloride (NED), L-cysteine and hydrochloric acid, ethylenediamine tetrakis (ethoxylate-block-propoxylate) tetrol (Tetronic 90R4), tween 20, tween 80, triton X-100, and sodium dodecyl sulfate (SDS) were of analytical grade and used as purchased from Sigma Aldrich (Saint Louis, MO, USA). The ultrapure water used for solution preparation was processed through a water purification system (Direct-Q ® 3, Millipore, Darmstadt, Germany) with resistivity equal to 18.2 MΩ cm at 25 ºC. Synthetic samples of normal and control serum were acquired from Doles (Goiânia, GO, Brazil) and Biotécnica (Varginha, MG, Brazil), respectively. Two blood plasma samples were received from the Laboratory of Clinical Analysis and Teaching in Health (LACES) from the Federal University of Goias.

Office paper (A4 size sheet) and adhesive tape (model 3M) were supplied by a local office supply store (Goiânia, GO, Brazil). The light emitting diode (LED) panel (dimension: 30  30  3.5 cm; 25 W), UV-LEDs and the printed circuit board were purchased from Embralumi (São Paulo, SP, Brazil) and Chipsce (São Paulo, SP, Brazil), respectively. The hollow cubic structure (dimensions: 25.5 cm (height)  28.2 cm (length)  27.0 cm (width)) composed by medium density fiberboard (MDF) and acrylonitrile butadiene styrene (ABS) thermoplastic filament (ø = 1.75 mm) were obtained from a local woodworking shop (Goiânia, GO, Brazil) and 3D Fila (Belo Horizonte, MG, Brazil), respectively.

Instrumentation

The 3D printer model Original Prusa i3 mk2/S, desktop scanner model G4050 and refrigerated micro centrifuge model NT 805 were purchased from Prusa Research (Prague, Czech Republic), Hewlett-Packard (Palo Alto, CA, United States) and Novatecnica (Piracicaba, SP, Brazil), respectively.

Fabrication of the mini studio

The mini studio was fabricated combining the following materials: MDF, office paper sheet and acrylic sheet. The MDF was utilized as the main structure of the holder. Four sheets of paper were fixed on each side and one acrylic sheet fixed on top of mini studio. Additionally, a LED panel was positioned on top of the mini studio at distance of ca. 13.5 cm. The assembling schematic representation of the mini studio segments is shown in Fig. S1 (Electronic Supplementary Material).

This system was combined with a Logitech 920 webcam (DropBot component) to capture the images of chip (Fig. S2).

Device Operation

The digital microfluidic operations were performed using the DropBot system model DB3-120 equipped with 90-pin array, high reflective chrome electrodes and a Logitech 920 webcam from SciBots (Kitchener, ON, Canada). The DMF devices comprised of 90 independent droplet actuation electrodes were connected via pogo pins to the open source DropBot control system. The movement of the droplets was controlled by MicroDrop software (version 3.1.3) and assays were performed using either real-time actuation mode or with semi-automated protocol. Droplet driving potentials ranged from 90 and 120 V, frequency of 10 kHz and time intervals ranging from 1 to 5 s. For loading and movement of droplets, the standard solution and biological samples were prepared in 0.5 mmol L -1 EDTA supplemented with 0.20% Tetronic 90R4 (v/v). The reservoirs were loaded with an aliquot (9 µL) of working solution and the droplets dispensed by activating a set of electrodes subsequently to the reservoir and deactivating the rectangular distribution electrode to generate a single unit droplet (~ 1 µL). The merging of two-unit droplets generated droplets of ~ 2 µL, as well as the division of one double-unit of droplet generated two single unit droplets. The droplet mixing process was performed by activating a circular layout of 4 electrodes mixed for ca. 30 s. The operation of dividing droplets from double to single unit was carried out by activating 3 sequential electrodes arranged linearly and later switching off the central electrode for 5 s. All DMF experiments were performed at room temperature (21 ± 2 ºC).

Fabrication of 3D printed holder

For the decomposition of CySNO directly on the chip surface, a 3D printed holder was manufactured and integrated with UV-LEDs. The device was designed by using SolidWorks software and additive manufactured through fused deposition modeling (FDM) for ~ 30 min. For this purpose, the filament ABS (black color) was used as additive and the object printed under the following infill conditions: layer height (0.2 mm), density (20%), temperatures at 240 ºC (nozzle) and 110 ºC (bed). Next, the parallel-connected array of four LEDs was integrated into the 3D holder. Fig. S3 illustrates the procedures involving the manufacturing step of the device and its assembling onto DMF chip.

Synthesis and decomposition of S-nitrosothiols

The CySNO was synthesized from an adaptation of the method reported by Peterson and coauthors [START_REF] Peterson | Decomposition of S-nitrosocysteine via S-to N-transnitrosation[END_REF]. The synthesis was carried out in the presence of an equimolar mixture of 50 mmol L -1 Lcysteine and nitrite (500 µL for each reagent) prepared in an aliquot of 2 mL of hydrochloric acid (0.1 mol L -1 ). After 5 min, the solution was diluted in 2 mL of a solution composed of 0.1 mol L -1 PBS (pH = 7.4), prepared in 0.5 mmol L -1 EDTA. The final concentration of CySNO was estimated by using UV-Vis spectrophotometry at 335 nm (ε = 736 M -1 cm -1 ). For the decomposition of CySNO groups on the DMF chip, an aliquot of standard sample (9 µL) was positioned on the reservoir region and exposed to UV light for 15 minutes.

On-chip colorimetric procedure

Two single unit droplets from different solutions, 50 mmol L -1 sulfanilamide prepared in 0.02% (v/v) Tetronic 90R4 and 4 mmol L -1 NED prepared in 0.01% (v/v) Tetronic 90R4 were dispensed from DMF reservoir electrode. Then, the droplets were merged, mixed and splited into other new single unit droplets of modified Griess reagent. Next, the droplet containing the Griess reagent was merged and mixed with a single unit droplet containing the solution of nitrite or decomposed CySNO (Figs. 1A-C). For evaluating the impact of polymeric surfactants on colorimetric responses, the type of polymeric surfactants and their concentration were optimized by using Griess reaction on circular plastic spots. On-chip parameters optimization was performed by two different experiments: (i) mixing configurations (including circular and linear procedures) and (ii) mixing time.

Analytical curves for nitrite and CySNO were done by using on-chip sequential dilutions from a 400 µmol L -1 stock concentration (either for nitrite or CySNO species). More information can be visualized in the Video S1 (available as supplemental). The repeatability and reproducibility of the semi-automatic protocol dedicated to the sample preparation, colorimetric reaction as well as the detection were successfully investigated based on the combination of real-time mode and preprogrammed operations.

Colorimetric detection

The on-chip colorimetric detection was monitored via image capturing with a high-resolution webcam (1280 x 720 pixels, frame rate = 10 FPS). The colorimetric analysis was performed in two simple steps: (i) the region of interest from colorimetric zones was captured by a print screen command and converted as PNG file format; (ii) the image colorimetric zone was analyzed by using Corel® PHOTO-PAINT™ X7 software, converting the color scale CMYK to magenta channel and collecting the pixel intensity (Fig. 1D). The pixel analysis used is like to other protocols reported in literature that successfully combine colorimetric paper-based devices with desktop scanner [START_REF] Cardoso | Colorimetric determination of nitrite in clinical, food and environmental samples using microfluidic devices stamped in paper platforms[END_REF][START_REF] De Oliveira | A simple method to produce 2D and 3D microfluidic paper-based analytical devices for clinical analysis[END_REF].

A schematic representation showing the chip design, reservoir sections, distribution of electrodes, actuation electrodes, colorimetric detection zones and the color capture process on zones are illustrated in Fig. S4. and image capture by print screen command and subsequent acquisition of pixel intensity using graphic design software.

Sample preparation

Two synthetic serum samples and two plasma samples were utilized as model biological matrices.

The above-mentioned samples were prepared by adding acetonitrile solution (dilution: 1:1 (v/v)) and centrifuging at 12000 rpm for 5 min. Next, the supernatant sample was diluted in a solution composed by 0.1 mol L -1 PBS (pH 7.4), 0.5 mmol L -1 EDTA and 0.2% Tetronic 90R4 (supernatant dilution: 1:1 (v/v)).

On-chip and reference analysis

For the comparative analysis, two different biological components were utilized: (i) synthetic blood serum and (ii) plasma samples. The experiment was done in the absence and presence of sample spiked with three different concentration levels of CySNO (25, 175 and 325 µmol L -1 ). For the DMF experiment, synthetic blood serum and plasma samples were loaded onto the chip reservoir.

Next, samples were decomposed under UV light, dispensed, and mixed with modified Griess reagent, and image was captured on device for CySNO analysis of pixel intensity. All DMF colorimetric assays were performed in quadruplicate.

In parallel experiments, the UV-Vis spectrophotometer model UV-M51 was purchased from BEL PHOTONICS (Piracicaba, SP, Brazil) and a calibration curve of nitrite was generated based on Griess-method (Fig. S5) [START_REF] Fox | Kinetics and Mechanisms of the Griess Reaction[END_REF][START_REF] Jayawardane | Microfluidic paper-based analytical device for the determination of nitrite and nitrate[END_REF]. Prior to spectrophotometric analysis, samples were submitted to the decomposition step. Next, the indirect detection of CySNO in the samples was achieved by combining aliquots of 1.5 mL of sample with 1.5 mL of Griess reagent in the quartz cuvette. Spectrophotometer responses were recorded at 510 nm (n=3).

Characterization

The performance of mini studio was characterized by four studies. (i) The background light responses from chip before and after the utilization of mini studio; (ii) the analysis of illuminance of mini studio, (iii) the comparison between the pixel intensity obtained on mini studio-webcam and desktop scanner responses and, (iv) the evaluation of the assembling of mini studio and DMF system yielded reproducible light quality results.

Background light results were collected with a print screen command of the chip in the absence and presence of mini studio. The analysis of luminosity was performed before starting the colorimetric analysis by using the smartphone application Lux Light Meter [START_REF] Park | Multifunctional hand-held sensor using electronic components embedded in smartphones for quick PCR screening[END_REF][START_REF] Gul | Multifunctional 3D-printed platform integrated with a smartphone ambient light sensor for halocarbon contaminants monitoring[END_REF]. This step was done by positioning the smartphone between the LED panel and the top of mini studio.

The comparative study involving the pixel capture was performed by collecting the pixel intensity directly on mini studio through a print screen command and by capturing the colorimetric zones through desktop scanner procedure [START_REF] Bhakta | Determination of nitrite in saliva using microfluidic paper-based analytical devices[END_REF][START_REF] Silva-Neto | Plug-and-play assembly of paper-based colorimetric and electrochemical devices for multiplexed detection of metals[END_REF]. These studies were used the Griess assay on polyester microwell plate [START_REF] Moreira | Fabrication of microwell plates and microfluidic devices in polyester films using a cutting printer[END_REF] as a model colorimetric reaction. Repeatability and reproducibility were calculated considering three sequential assembling stages of mini studio and analyzing the background signals directly collected on DMF chip.

RESULTS AND DISCUSSION

Integration of colorimetric detector on chip

DMF has been successfully explored in numerous cases involving chemical and bioanalytical applications due to its benefits as a programmable droplet manipulation tool, full sample preparation capabilities, portability, and potential to be integrated with different detection approaches [START_REF] Samiei | A review of digital microfluidics as portable platforms for lab-on a-chip applications[END_REF][START_REF] Choi | Digital microfluidics[END_REF][START_REF] Jebrail | Digital microfluidics: A versatile tool for applications in chemistry, biology and medicine[END_REF]. However, the most popular examples reported in the literature do not explore the colorimetric analysis without laborious mathematics pre-treatment. In addition, the control of background lighting induced auto-reflection patterns on the metallic surfaces of the DMF electrodes can be challenger for reproducible optical analysis [START_REF] Kalsi | A programmable digital microfluidic assay for the simultaneous detection of multiple anti-microbial resistance genes[END_REF][START_REF] Gu | Integrated Digital Microfluidic Platform for Colorimetric Sensing of Nitrite[END_REF][START_REF] Jafry | Double-sided electrohydrodynamic jet printing of two-dimensional electrode array in paper-based digital microfluidics[END_REF][START_REF] Yafia | Ultra-portable smartphone controlled integrated digital microfluidic system in a 3D-printed modular assembly[END_REF][START_REF] Gu | Colorimetric sensing with gold nanoparticles on electrowetting-based digital microfluidics[END_REF][START_REF] Huang | Quantitative measurements of inorganic analytes on a digital microfluidics platform[END_REF].

In view of the recent studies, the strategy to mitigate the inconveniences can be solved through manufacturing an alternative mini studio and combining the luminosity control approach inside the studio with the subsequent capture of the pixel responses from colorimetric product formed directly on a chip using a basic print screen command.

Fabrication and characterization of mini studio

The proposed strategy exploiting a mini studio was evaluated to control the background light on the DMF chip during the colorimetric experiments. A luminosity control tool was constructed based on combination of four "do it yourself" materials. First, a cubic structure composed by MDF containing open faces was designed. The side faces of the MDF structure were assembled with three sheets of paper, while the top face of the cube housed one sheet of white acrylic. Finally, a LED panel was successfully positioned at 13.5 cm from the top of mini studio. We decided to insert the acrylic structure on top of mini studio to ensure homogenous light diffusion from the LED panel.

To examine the performance of the mini studio, the DMF system was positioned inside of proposed tool and three different experiments were carefully evaluated. One study was carried out by collecting the background light responses from the chip in the absence of mini studio and through the usage of a mini studio with five different LED configurations. As indicated in Fig. 2, the studied modes include the use of (i) Luminaires with 36 LEDs, (ii) 20 circular LEDs inside the mini studio, (iii) 60 LEDs, (iv) 30 LEDs and (v) panel LED. The choice of the most promising strategy for lighting control was selected based on the pixel intensity values of the 30 central actuation electrodes.

The second experiment was based on illuminance measurements collected between the LED panel and top of mini studio (Table S1). Finally, the third characterization was performed comparing the colorimetric responses on proposed system with results obtained by digitalization on desktop scanner (Figs. S6 A-C). The desktop scanner colorimetric experiments were performed on a polyester microwell plate [START_REF] Moreira | Fabrication of microwell plates and microfluidic devices in polyester films using a cutting printer[END_REF] since the chrome DMF chip could not be scanned to be used as a comparison parameter due to its high reflection. The results seen in Fig. 2 represent the luminosity on the surface of the DMF chip before and after the utilization of mini studio and their background light results obtained through magenta color channel. The pixel responses observed in the absence of mini studio indicated poor homogeneity of background signal, with pixel intensity value of 102 ± 52 AU. This behavior can be associated with the reflection of electrodes present on the DMF chip (see Fig. 2A). As indicated in Figs. 2B-F, the presence of mini studio integrated with different LEDs revealed gradual reduction of background light and improvement of pixel homogeneity, with pixel intensity responses ranged between 63 ± 11 and 3.1 ± 0.3. The best background light condition was observed when the mini studio was integrated with a LED panel, which demonstrated a decrease in the background signal in terms of pixel intensity in ~ 97% when compared to the background signal in the absence of mini studio (Fig. 2G). Further information related to the proposed mini studio can be seen in Fig. S2.

It is important to mention that the proposed approach combining the mini studio, LED panel and webcam can promote acceptable background light control, thus emerging as affordable alternative for monitoring colorimetric reactions on-chip. In addition, the adapted optical sensor is an auxiliary component from the DropBot system that allows a direct connection to the MicroDrop software, making possible any adjustments involving the image capture process.

Table S1 shows the obtained values of light intensity density with the mini studio. The illuminance analysis indicated a light intensity density value of ~ 12689 ± 545 Lux (n = 3). This qualitative measurement exhibited reproducible results, indicating high homogeneity of light emission from the LED panel. Thus, the simple combination of DMF system and the mini studio can solve two important challenges in optical sensing: strong variations in the background signal from chip and erroneous interpretations of the color responses extracted from the colorimetric assays due to background variation. In addition, the proposed system has ensured great simplicity in the collection of the analytical signal and dispenses additional data treatments.

Figs. S6A-B demonstrate the colorimetric responses obtained from desktop scanner in comparison

to the mini studio integrated with webcam. Based on each analytical curve, the sensitivity values achieved using scanner and mini studio were 0.252 and 0.215 AU/µmol L -1 , respectively. The better sensitivity performance observed upon the desktop scanner can be associated to the fact that it presents a robust control of luminosity. However, the required time to digitalization the colorimetric zones via desktop scanner was ~ 5 min. On the other hand, the mini studio assembled with webcam provided similar background light noise and faster digital image acquisition (~ 5 s). The statistical correlation between the responses obtained with the mini studio system versus the desktop scanner method was also evaluated (Fig. S6C). The calculated correlation value was 0.9, which suggests high correlation between both methods. Thus, the proposed mini studio (when combined with webcam tool) can promote a favorable and simple process for digitalization the colorimetric zones on-chip. Considering the faster time for performing all colorimetric assay with the print screen function acquisition, the proposed tool has great potential for monitoring the kinetic colorimetric reactions.

To evaluate the repeatability and reproducibility of the light control conditions when assembling the mini studio and DMF system together, three procedures in sequence were performed. The chip surface images were acquired after each assembly and the relative standard deviation (RSD) values from background light noise of the 30 central actuation electrodes were calculated for each experiment. The obtained results of pixel intensity were submitted to one-way analysis of variance (ANOVA; p = 0.05). The results summarizing the captured zones and their respective histograms are shown in Figs. S7A-D. The calculated values of RSD for repeatability and reproducibility ranged from 4 and 2%, respectively, thus indicating satisfactory performance. Furthermore, through the ANOVA analysis, a F test was performed, and the obtained value (F = 2.14) was lower than the critical F value (F crit 3.10), indicating absence of statistical difference between the three different assembling processes at 95% confidence level. These results suggest that the procedure of colorimetric analysis can be performed in a large space of the chip, discarding the need to move the colorimetric droplet to the specific point on the chip.

Optimization of operational parameters

The utilization of surfactants in DMF assays facilitates the actuation of droplets, improving the sampling and reducing possible biofouling on electrode surfaces [START_REF] De Campos | plug-n-Play" Sensing with Digital Microfluidics[END_REF][START_REF] Kirby | Digital microfluidics: An emerging sample preparation platform for mass spectrometry[END_REF][START_REF] Raccurt | On the influence of surfactants in electrowetting systems[END_REF][START_REF] Ng | A digital microfluidic system for serological immunoassays in remote settings[END_REF]. To evaluate the effects of surfactant on colorimetric Griess reaction responses, a set up experiments was carried out in the absence and presence of following surfactants: Tetronic 90R4, triton-X 100, tween 20, tween 80 and sodium dodecyl sulfate (SDS). Colorimetric signal was captured between 0 and 30 min after starting the Griess reaction. Additionally, the concentration of the selected above polymeric surfactant was optimized with prepared levels ranging from 0.001 to 0.2% (v/v).

As can be seen in Fig. S8, colorimetric zones in the absence and presence of surfactants did not show significant difference in terms of pixel intensity, which values ranged from 23 to 28 AU. This result indicates that the presence of surfactants does not alter significantly the signal intensity for the detection of nitrite. In addition, the utilization of Tetronic 90R4 as polymeric surfactant exhibited the best colorimetric response. The best chemical conditions for the Griess assay on-chip were obtained when the solutions of sulfanilamide, NED and nitrite were supplemented with Tetronic 90R4 at concentrations 0.02, 0.01 and 0.20% (v/v), respectively. Additional results are shown in Table S2.

The effect of different types of droplets mixing on-chip was also investigated through both circular and linear mixing configurations. A study reported by Li et al. [START_REF] Li | Intelligent droplet tracking with correlation filters for digital microfluidics[END_REF] using the circular droplet mixing pattern was employed to assess the reaction kinetics. In their study, the authors used droplets of ca.

15 µL to assess the colorimetric response after incubation times between 1 and 10 minutes.

In this current report, optimizations for the droplet mixing type and time using the colorimetric Griess reaction were performed using a droplet of 200 μmol L -1 NO 2 -in 0.1 mol L -1 PBS (pH = 7.4), 0.5 mmol L -1 EDTA and 0.20% Tetronic (v/v) and a droplet of prepared Griess reagent. Driving potential was 100 V and, mixing time ranged from 1 to 60 s, as summarized in Fig. 3. The observed behavior in both droplet mixing processes did not provide noticeable differences on colorimetric results, which calculated values of pixel intensity of 27.7 ± 0.4 and 27 ± 1 AU, for circular mixing and linear mixing strategies, respectively (see Figs. 3A-B). However, the mixture process via circular approach exhibited lower RSD values and, consequently, it was selected as ideal operation condition. The results displayed in Figs. 3C-D indicate that the time of 15 s is insufficient to promote a good diffusibility for mixing between NO 2 -and Griess reagents. On the other hand, the best droplet mixing efficiency was acquired at ~ 30 s. It was also observed that for longer mixing procedures (t > 60 s), droplets may be susceptible to decrease in color intensity formed on-chip.

On-chip CySNO decomposition

The Saville method is one of the main approaches utilized in literature for the decomposition of Snitrosothiols and their indirect colorimetric detection [START_REF] Saville | A scheme for the colorimetric determination of microgram amounts of thiols[END_REF]. This procedure is based on the use of mercury(II) chloride as catalyst material to convert S-nitrosothiols groups in nitrite and other subproducts. Subsequently, the nitrite can be measured by using the combination of Griess reaction and spectrophotometric techniques. Other studies successfully reported the use of toxic reagent free methods, such as UV radiation exposure, for promoting S-nitrosothiols degradation with similar results [START_REF] Aravindakumar | Decomposition of s-nitrosothiols induced by UV and sunlight[END_REF][START_REF] Hunter | S-Nitrosothiol Analysis via Photolysis and Amperometric Nitric Oxide Detection in a Microfluidic Device[END_REF].

In this study, we propose the degradation of S-nitrosothiols directly on DMF chips using the reagent-free approach by combining a customized UV-3D printed holder coupled with DMF apparatus. The proposed plastic holder uses UV-LEDs mounted on a 3D printed support that can be positioned on top of the DMF device. Aiming for best performance, the on-chip degradation of CySNO group was optimized through five different experiments ranging the degradation time from 0 to 15 min. Fig. S9 illustrates the real image of system used UV-LED in combination with DMF platform. The observed percentage of CySNO decomposition into NO 2 -through the homolytic cleavage via UV exposure was 7, 45, 54, 78, and 96% for times 0, 2.5, 5, 10 and 15 min, respectively. Thus, the degradation time of 15 min yielded the best condition for performing the reagent-free on-chip degradation process of CySNO groups (see Figs. 4A-B), which is in good agreement with previously reported studies [START_REF] Ismail | Colorimetric analysis of the decomposition of S-nitrosothiols on paper-based microfluidic devices[END_REF][START_REF] Tyurin | Elevated levels of S-nitrosoalbumin in preeclampsia plasma[END_REF]. In the above-mentioned condition, the humidity control in the environment is recommended to prevent evaporation processes, as described by other authors [START_REF] Berthier | Managing evaporation for more robust microscale assays: Part 1. Volume loss in high throughput assays[END_REF][START_REF] Nelson | Incubated protein reduction and digestion on an electrowetting-on-dielectric digital microfluidic chip for MALDI-MS[END_REF]. UV degradation times longer than 15 min should be avoided (for on chip experiments), deviation of the expected degradation results could happen as result of the droplet evaporation on device. An alternative to improve this effect could be the introduction of an immiscible fluid system through use of oil to prevent droplet evaporation [START_REF] Hong | Three-dimensional digital microfluidic manipulation of droplets in oil medium[END_REF][START_REF] Cheng | Recent advances in magnetic digital microfluidic platforms[END_REF].

Sensing performance

Two different analytical curves were constructed by loading the Griess and target analyte solutions on chip under optimized experimental conditions. One analytical plot was performed using standard solutions of nitrite while the second was obtained with CySNO. Both analytical curves were acquired with standard solutions prepared in the concentration range from 12.5 to 400 µmol L -1 (Fig. 4 C-D). The linear equations were y = (0.152 ± 0.004)x -(1.8 ± 0.3) with R 2 = 0.998 and y = (0.141 ± 0.003)x -(0.4 ± 0.4) with R 2 = 0.997 for the responses of NO 2 -and CySNO samples, respectively. The limit of detection (LOD) was calculated based on 3*SD blank /slope (n = 10). The calculated LOD values for both NO 2 -and CySNO were 2.8 µmol L -1 . As showed in Table S3, the analytical parameters exhibited similar performance when compared to other results reported in literature. The repeatability and reproducibility of the semi-automatic protocol on chip involving the detection of NO 2 -and CySNO were calculated based on pixel intensity responses (one device with n = 4 for repeatability; two devices with n = 8 for reproducibility). In terms of repeatability, the respective RSD values involving the detection of NO 2 -and CySNO were 2 and 4%, respectively. Considering the reproducibility study, the found values ranged from 5 and 8%. It is important to mention that, to the best of our knowledge, this is the first report that uses the combination of mini studio and DMF system for in-situ degradation and detection of CySNO on-chip.

Practical applicability and reference analysis

As proof of concept, the proposed semi-automatic method was selected for the UV-decomposition, reagent prepration and indirect detection of CySNO in four biological samples (two control serum and other two blood plasma samples). Aiming to validate the proposed method, two distinct accuracy approaches were carefully explored by using synthetic and blood serum samples: recovery study and by using a reference analysis (spectrophotometric technique). The biological solutions were spiked with CySNO standard solutions prepared at three concentration levels: 25, 175 and 325 µmol L -1 . Table 1 summarizes the obtained analytical responses using the DMF and spectrophotometry. [START_REF] De Campos | plug-n-Play" Sensing with Digital Microfluidics[END_REF] for the data recorded using serum and plasma as samples were lower than the critical t value (2.91), thus indicating that the results did not differ significantly at a 95% confidence level. Based on the achieved results, the proposed approach has provided satisfactory accuracy through a semi-automatic protocol for performing the colorimetric analysis. Finally, two control serum samples and two blood plasma samples were analyzed before and after degradation step. Fig. 5 shows the obtained images for the tested samples and the obtained nitrite concentrations before and after CySNO degradation are presented in Table 2. As displayed in Table 2, the measured level of nitrite in biological samples (#1 to #4) ranged from 18 to 27 µmol L -1 and the indirect analysis of CySNO in samples #4 was 6 ± 1 µmol L -1 . It is important to mention that the nitrite response (pre irradiation) was subtracted from the RSNO response (post irradiation). In addition, the concentration of CySNO was expressed in terms of lowmolecular weight S-nitrosothiol. Unfortunately, the levels of CySNO in samples #1, #2 and 3# were below the LOD and their concentrations were not quantified. The assembling of DMF chips into a mini studio containing a 3D printed holder with integrated LEDs has successfully demonstrated a huge potential to promote a complete analysis including the on-chip degradation of the biological compound, which can be useful for assessing quantitative information on the concentration of target and clinically relevant compounds on biological fluids.

CONCLUSIONS

This study has described the assembling and integration of DMF platforms into a mini studio for controlling the background light during colorimetric measurements, thus enhancing the ability of digital microfluidics to perform reproducible complete analysis with sample-in-answer-out capability. The strategy adopted herein comprised the integration of 3D printed holder containing previously fixed UV LEDs to promote for the first time the degradation of low molecular weight Snitrosothiols directly on DMF chips prior to promote a complete analytical procedure through a semi-automated protocol. The proposed approach offered great performance for real sample analyzes with no statistical difference from the results recorded through a reference technique.

Lastly, it is important to highlight that the integration of UV degradation directly on DMF chip followed by reagent mixing, colorimetric reaction and signal readout opens a new gate to be explored in analytical and bioanalytical chemistry including, but not limited, the analysis of other target compounds potentially relevant for clinical diagnostics.

CRediT authorship contribution statement

Fig. 1 .

 1 Fig. 1. (A) DMF and mini studio components. (B) Integration between 3D printed UV camera and DMF platform. (C) Droplet generation, fluid arrangement system and decomposition of CySNO by UV radiation and mixture of the degradation product with the chromogen (modified Griess reagent) and image capture by print screen command and subsequent acquisition of pixel intensity using graphic design software.

Fig. 2 .

 2 Fig. 2. Strategies for luminosity control on the DMF chip. (A) Absence of studio. (B) Use of "do-ityourself" mini studio. (C) Mini studio associated with 20 internal LEDs. (D) Mini studio associated with a circular array of 36 LEDs. (E) Mini studio coupled to 60 rectangular LEDs. (F) Mini studio coupled to a rectangular array of 30 LEDs. (G) Mini studio combined with ceiling light. (H) Histogram of the average pixel intensities of the different lighting strategies (n = 30).

Fig. 3 .

 3 Fig. 3. Mixing type and time of mixing using 200 μmol L -1 NO 2 -in 0.1 mol L -1 PBS (pH = 7.4), 0.5 mmol L -1 EDTA and 0.20% Tetronic (v/v) after reaction with Griess reagent. (A) Magenta color product of the reaction in the colorimetric detection zones on the DMF chip after assays utilizing circular and linear mixing. (B) Histogram of pixel intensity obtained in the automated mixing type optimization test (n = 4). (C) Magenta reaction product in the colorimetric detection zone after different times of automated mixing. (D) Histogram of pixel intensities obtained at different mixing times (n = 4).

Fig. 4 .

 4 Fig. 4. (A) Colorimetric response of 400 µmol L -1 CysNO prepared in 0.1 mol L -1 PBS (pH = 7.4), 0.5 mmol L -1 EDTA and 0.20% Tetronic 90R4 (w/v) before and after degradation in different times on the DMF device. (B) Pixel intensity as a function of CySNO degradation time (n = 4). (C) Concentration gradient obtained in the construction of the analytical curve of NO 2 -and CySNO on the digital microfluidics platform. (D) Analytical curve obtained for NO 2 -and CySNO (after degradation with UV light) in 0.1 mol L -1 PBS, 0.5 mmol L -1 EDTA and 0.20% Tetronic (v/v) using DMF (n = 4).

Fig. 5 .

 5 Fig. 5. DMF images of control serum and blood plasma samples before and after degradation with subsequent reaction with Griess agent.

Table 1

 1 Comparison between three level CySNO addition and recovery assay for artificial serum and blood plasma samples using DMF and spectrophotometry techniques.As shown in Table1, the recovery values for DMF tests ranged from 78 to 104% and 80 to 100% for the control se-rum and blood plasma samples, respectively. In addition, the results presented in Table1demonstrate that analysis of CySNO through DMF and reference technique

	Sample	Spiking level	Added (µmol L -1 )	(µmol L -1 )	DMF Recovery (%)	Spectrophotometry (µmol L -1 ) Recovery (%)
	Serum	#1	25	26 ± 4	104	21 ± 1	84
	Serum	#2	175	148 ± 4	84	160 ± 1	91
	Serum	#3	325	255 ± 4	78	278 ± 6	86
	Plasma	#4	25	25 ± 9	100	25 ± 1	100
	Plasma	#5	175	140 ± 9	80	149 ± 9	85
	Plasma	#6	325	297 ± 18	91	255 ± 7	78

Table 2

 2 NO 2 -and CySNO values found in control serum and blood plasma diluted samples.

	Sample	Nitrite (µmol L -1 )	LMW-RSNO (µmol L -1 )
	#1	18 ± 4	< LOD
	#2	23 ± 6	< LOD
	#3	20 ± 3	< LOD
	#4	27 ± 7	6 ± 1

Acknowledgments

The authors would like to thank CAPES (finance code 88887.192880/2018-00 and 001), CNPq (grants 307554/2020-1 and 405620/2021-7) and INCTBio (grant 465389/2014-7) for the financial support and granted scholarships. The Laboratory of Clinical Analysis and Health Education (LACES) and Professor Paulo Sérgio de Souza are also acknowledged for donating blood plasma samples for DMF analysis and technical supporting for spectrophotometric analysis, respectively. Lastly, the authors would like to thank Professor Aaron Wheeler for suggestions, discussions, and support to implement DMF in our group.

Declaration of competing interest

The authors declare no competing financial interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: Experimental optimizations, characterizations, and real illustrations of the proposed system (PDF).

A semi-automatic analytical procedure video is also supplied in separate file (MP4).