Quantum dots-based "chemical tongue" for the discrimination of short-length Aß peptides.

A "chemical tongue" is proposed based on thiomalic acid-capped quantum dots (QDs) with signal enrichment provided by excitation-emission matrix (EEM) fluorescence spectroscopy for the determination of close structural analogs-short-length amyloid ß (Aß) peptides related to Alzheimer's disease. Excellent discrimination is obtained by principal component analysis (PCA) for seven derivatives: Aß1-16, Aß4-16, Aß4-9, Aß5-16, Aß5-12, Aß5-9, Aß12-16. Detection of Aß4-16, Aß4-16, and Aß5-9 in binary and ternary mixtures performed by QDs-based chemical tongue using partial least squares-discriminant analysis (PLS-DA) provided perfect 100% accuracy for the two studied peptides (Aß4-16 and Aß4-16), while for the third one (Aß5-9) it was slightly lower (97.9%). Successful detection of Aß4-16 at 1 pmol/mL (1.6 ng/mL) suggests that the detection limit of the proposed method for short-length Aß peptides can span nanomolar concentrations. This result is highly promising for the development of simple and efficient methods for sequence recognition in short-length peptides and better understanding of mechanisms at the QD-analyte interface.

Portable Surface Plasmon Resonance Detector for COVID-19 Infection.

Methods based on nucleic acid detection are currently the most commonly used technique in COVID-19 diagnostics. Although generally considered adequate, these methods are characterised by quite a long time-to-result and the necessity to prepare the material taken from the examined person-RNA isolation. For this reason, new detection methods are being sought, especially those characterised by the high speed of the analysis process from the moment of sampling to the result. Currently, serological methods of detecting antibodies against the virus in the patient's blood plasma have attracted much attention. Although they are less precise in determining the current infection, such methods shorten the analysis time to several minutes, making it possible to consider them a promising method for screening tests in people with suspected infection. The described study investigated the feasibility of a surface plasmon resonance (SPR)-based detection system for on-site COVID-19 diagnostics. A simple-to-use portable device was proposed for the fast detection of anti-SARS-CoV-2 antibodies in human plasma. SARS-CoV-2-positive and -negative patient blood plasma samples were investigated and compared with the ELISA test. The receptor-binding domain (RBD) of spike protein from SARS-CoV-2 was selected as a binding molecule for the study. Then, the process of antibody detection using this peptide was examined under laboratory conditions on a commercially available SPR device. The portable device was prepared and tested on plasma samples from humans. The results were compared with those obtained in the same patients using the reference diagnostic method. The detection system is effective in the detection of anti-SARS-CoV-2 with the detection limit of 40 ng/mL. It was shown that it is a portable device that can correctly examine human plasma samples within a 10 min timeframe.

Study on Saccharide-Glucose Receptor Interactions with the Use of Surface Plasmon Resonance.

The aim of this study was to investigate the process of attachment of saccharide particles differing in degree of complexity to cell receptors responsible for transport of glucose across the cell membrane (GLUT proteins). This phenomenon is currently considered when designing modern medicines, e.g., peptide drugs to which glucose residues are attached, enabling drugs to cross the barrier of cell membranes and act inside cells. This study aims to help us understand the process of assimilation of polysaccharide nanoparticles by tumour cells. In this study, the interactions between simple saccharides (glucose and sucrose) and dextran nanoparticles with two species of GLUT proteins (GLUT1 and GLUT4) were measured using the surface plasmon resonance technique. We managed to observe the interactions of glucose and sucrose with both applied proteins. The lowest concentration that resulted in the detection of interaction was 4 mM of glucose on GLUT1. Nanoparticles were measured using the same proteins with a detection limit of 40 mM. These results indicate that polysaccharide nanoparticles interact with GLUT proteins. The measured strengths of interactions differ between proteins; thus, this study can suggest which protein is preferable when considering it as a mean of nanoparticle carrier transport.

Targeted Delivery of Cisplatin by Gold Nanoparticles: The Influence of Nanocarrier Surface Modification Type on the Efficiency of Drug Binding Examined by CE-ICP-MS/MS.

Spherical gold nanoparticles (GNPs), whose unique properties regarding biomedical applications were broadly investigated, are an object of interest as nanocarriers in drug targeted delivery systems (DTDSs). The possibility of surface functionalization, especially in enabling longer half-life in the bloodstream and enhancing cellular uptake, provides an opportunity to overcome the limitations of popular anticancer drugs (such as cisplatin) that cause severe side effects due to their nonselective transportation. Herein, we present investigations of gold nanoparticle-cisplatin systems formation (regarding reaction kinetics and equilibrium) in which it was proved that the formation efficiency and stability strongly depend on the nanoparticle surface functionalization. In this study, the capillary electrophoresis hyphenated with inductively coupled plasma tandem mass spectrometry (CE-ICP-MS/MS) was used for the first time to monitor gold-drug nanoconjugates formation. The research included optimizing CE separation conditions and determining reaction kinetics using the CE-ICP-MS/MS developed method. To characterize nanocarriers and portray changes in their physicochemical properties induced by the surface's processes, additional hydrodynamic size and ζ-potential by dynamic light scattering (DLS) measurements were carried out. The examinations of three types of functionalized GNPs (GNP-PEG-COOH, GNP-PEG-OCH3, and GNP-PEG-biotin) distinguished the essential differences in drug binding efficiency and nanoconjugate stability.

Comparative Evaluation of Different Surface Coatings of Fe3O4-Based Magnetic Nano Sorbent for Applications in the Nucleic Acids Extraction.

Nucleic acid extraction and purification are crucial steps in sample preparation for multiple diagnostic procedures. Routine methodologies of DNA isolation require benchtop equipment (e.g., centrifuges) and labor-intensive steps. Magnetic nanoparticles (MNPs) as solid-phase sorbents could simplify this procedure. A wide range of surface coatings employs various molecular interactions between dsDNA and magnetic nano-sorbents. However, a reliable, comparative evaluation of their performance is complex. In this work, selected Fe3O4 modifications, i.e., polyethyleneimine, gold, silica, and graphene derivatives, were comprehensively evaluated for applications in dsDNA extraction. A family of single batch nanoparticles was compared in terms of morphology (STEM), composition (ICP-MS/MS and elemental analysis), surface coating (UV-Vis, TGA, FTIR), and MNP charge (ζ-potential). ICP-MS/MS was also used to unify MNPs concentration allowing a reliable assessment of individual coatings on DNA extraction. Moreover, studies on adsorption medium (monovalent vs. divalent ions) and extraction buffer composition were carried out. As a result, essential relationships between nanoparticle coatings and DNA adsorption efficiencies have been noticed. Fe3O4@PEI MNPs turned out to be the most efficient nano sorbents. The optimized composition of the extraction buffer (medium containing 0.1 mM EDTA) helped avoid problems with Fe3+ stripping, which improved the validity of the spectroscopic determination of DNA recovery.

Versatile and Easily Designable Polyester-Laser Toner Interfaces for Site-Oriented Adsorption of Antibodies.

Laser toners appear as attractive materials for barriers and easily laminated interphases for Lab-on-a-Foil microfluidics, due to the excellent adhesion to paper and various membranes or foils. This work shows for the first time a comprehensive study on the adsorption of antibodies on toner-covered poly(ethylene terephthalate) (PET@toner) substrates, together with assessment of such platforms in rapid prototyping of disposable microdevices and microarrays for immunodiagnostics. In the framework of presented research, the surface properties and antibody binding capacity of PET substrates with varying levels of toner coverage (0-100%) were characterized in detail. It was proven that polystyrene-acrylate copolymer-based toner offers higher antibody adsorption efficiency compared with unmodified polystyrene and PET as well as faster adsorption kinetics. Comparative studies of the influence of pH on the effectiveness of antibodies immobilization as well as measurements of surface ζ-potential of PET, toner, and polystyrene confirmed the dominant role of hydrophobic interactions in adsorption mechanism. The applicability of PET@toner substrates as removable masks for protection of foil against permanent hydrophilization was also shown. It opens up the possibility of precise tuning of wettability and antibody binding capacity. Therefore, PET@toner foils are presented as useful platforms in the construction of immunoarrays or components of microfluidic systems.

Red Flags and Adversities on the Way to the Robust CE-ICP-MS/MS Quantitative Monitoring of Self-Synthesized Magnetic Iron Oxide(II, III)-Based Nanoparticle Interactions with Human Serum Proteins.

The growing interest in superparamagnetic iron oxide nanoparticles (SPIONs) as potential theranostic agents is related to their unique properties and the broad range of possibilities for their surface functionalization. However, despite the rapidly expanding list of novel SPIONs with potential biomedical applications, there is still a lack of methodologies that would allow in-depth investigation of the interactions of those nanoparticles with biological compounds in human serum. Herein, we present attempts to employ capillary electrophoresis-inductively coupled plasma tandem mass spectrometry (CE-ICP-MS/MS) for this purpose and various obstacles and limitations noticed during the research. The CE and ICP-MS/MS parameters were optimized, and the developed method was used to study the interactions of two different proteins (albumin and transferrin) with various synthesized SPIONs. While the satisfactory resolution between proteins was obtained and the method was applied to examine individual reagents, it was revealed that the conjugates formed during the incubation of the proteins with SPIONs were not stable under the conditions of electrophoretic separation.

Nanoparticles of chosen noble metals as reactive oxygen species scavengers.

Reactive oxygen species (ROS) play an important role in various physiological processes of living organisms. However, their increased concentration is usually considered as a threat for our health. Plants, invertebrates, and vertebrates including humans have various enzymatic and non-enzymatic defence systems against ROS. Unfortunately, both bad condition of surrounding environment and unhealthy lifestyle can interfere with an activity of enzymes responsible for a regulation of ROS levels. Therefore, it is important to look for alternative ROS scavengers, which could be administrated to chosen tissues to prevent pathological processes such as distortion of DNA or RNA structures and oxidation of proteins and lipids. One of the most recently proposed solutions is the application of nanozymes, which could mimic the activity of essential enzymes and prevent excessive activity of ROS. In this work, nanoparticles of Au, Pt, Pd, Ru and Rh were synthesized and studied in this regard. Peroxidase-, catalase (CAT)- and superoxide dismutase (SOD)-like activity of obtained nanoparticles were tested and compared using different methods. The influence of bovine and human albumins on CAT- and peroxidase-like activity was examined. Moreover, in the case of CAT-like activity, an influence of pH and temperature was examined and compared. Determination of SOD-like activity using the methods described for the examination of the activity of native enzyme was not fully successful. Moreover, cytotoxicity of chosen nanoparticles was studied on both regular and tumor cells.

Excitation-emission fluorescence matrix acquired from glutathione capped CdSeS/ZnS quantum dots in combination with chemometric tools for pattern-based sensing of neurotransmitters.

The presented work concerns pattern-based sensing with quantum dots for the identification and quantification of neurotransmitters by means of excitation-emission fluorescence spectroscopy (2D fluorescence). In the framework of this study, glutathione capped CdSeS/ZnS nanocrystals were used as non-specific nanoreceptors capable of differentiated interaction with neurotransmitters. The pattern-based sensing with QDs was realized by using excitation-emission fluorescence spectroscopy to provide analyte-specific multidimensional optical information. These characteristic fluorescent response patterns were processed by unfolded partial least squares-discriminant analysis, showing that satisfactory identification of all investigated neurotransmitters: dopamine, norepinephrine, epinephrine, serotonin, GABA, and acetylcholine, can be achieved through the proposed sensing strategy. The impact of the considered fluorescence signal (datum, i.e. zeroth-order data acquired per sample; spectrum, i.e. first-order data acquired per sample; excitation-emission matrix, i.e. second-order data acquired per sample) on the sensing capability of glutathione capped QDs was also verified. The best performance parameters such as accuracy, precision, sensitivity, and specificity were obtained using excitation-emission matrices (88.9-93.3%, 0.93-0.95, 0.89-0.93, and 0.99-1.00, respectively). Thus, it was revealed that excitation-emission fluorescence spectroscopy may improve the recognition of neurotransmitters while using only one type of nanoreceptor. Furthermore, is was demonstrated that the proposed excitation-emission fluorescence spectroscopy assisted QD assay coupled with unfolded partial least squares regression can be successfully utilized for quantitative determination of catecholamine neurotransmitters at the micromolar concentration range with R2 in the range 0.916-0.987. Consequently, the proposed sensing strategy has the potential to significantly simplify the sensing element and to expand the pool of bioanalytes so far detectable with the use of QDs.

Recent Advancements in Receptor Layer Engineering for Applications in SPR-Based Immunodiagnostics.

The rapid progress in the development of surface plasmon resonance-based immunosensing platforms offers wide application possibilities in medical diagnostics as a label-free alternative to enzyme immunoassays. The early diagnosis of diseases or metabolic changes through the detection of biomarkers in body fluids requires methods characterized by a very good sensitivity and selectivity. In the case of the SPR technique, as well as other surface-sensitive detection strategies, the quality of the transducer-immunoreceptor interphase is crucial for maintaining the analytical reliability of an assay. In this work, an overview of general approaches to the design of functional SPR-immunoassays is presented. It covers both immunosensors, the design of which utilizes well-known and often commercially available substrates, as well as the latest solutions developed in-house. Various approaches employing chemical and passive binding, affinity-based antibody immobilization, and the introduction of nanomaterial-based surfaces are discussed. The essence of their influence on the improvement of the main analytical parameters of a given immunosensor is explained. Particular attention is paid to solutions compatible with the latest trends in the development of label-free immunosensors, such as platforms dedicated to real-time monitoring in a quasi-continuous mode, the use of in situ-generated receptor layers (elimination of the regeneration step), and biosensors using recombinant and labelled protein receptors.

Monitoring of iron status in patients with heart failure.

The 2016 ESC/HFA heart failure (HF) guidelines emphasize the importance of identifying and treating iron deficiency (ID) in patients with HF. Iron deficiency can occur in half or more of HF sufferers, depending on age and the phase of the disease. Iron deficiency can be a cause of anaemia, but it is also common even without anaemia, meaning that ID is a separate entity, which should be screened for within the HF population. Although assessment of iron stores in bone marrow samples is the most accurate method to investigate iron status, it is not practical in most HF patients. Levels of circulating iron biomarkers are an easily available alternative; especially, ferritin and transferrin saturation (Tsat). In patients with HF serum ferritin level <100 µg/L (regardless of Tsat value) or between 100 and 299 µg/L with Tsat <20% are considered as recommended criteria for the diagnosis of ID, criteria which have been used in the clinical trials in HF that have led to a recommendation to treat ID with intravenous iron. We discuss the optimal measures of iron biomarkers in patients with HF in order to screen and monitor iron status and introduce some novel ways to assess iron status.

Drug therapy in elderly heart failure patients.

Although heart failure (HF) is considered as a cardiogeriatric syndrome, elderly and very elderly patients are under-represented in the vast majority of clinical trials investigating novel drugs and therapies in this population. The homoeostatic systems of elderly subjects are very fragile, and the management of HF accompanied by numerous comorbidities requires a holistic approach towards the patient, with special emphasis not only on psychosomatic problems but also on the individual (including social) needs of each particular patient, along with the support for the family and/or caregivers. In this article, we summarize current evidence regarding pharmacotherapy of elderly patients with HF and summarize the clinical problems occurring in this population.

Iron Deficiency Treatment in Patients with Heart Failure.

Iron deficiency (ID) is one of the major risk factors for disability and mortality worldwide, and it was identified as a common and ominous comorbidity in patients with heart failure (HF), both with and without anaemia. Based on two clinical trials (FAIR-HF and CONFIRM-HF) and other epidemiological evidence, ID has been recognized as an important therapeutic target in symptomatic patients with HF and LVEF ≤45%.Intravenous iron supplementation has been demonstrated to be safe and effective for iron repletion and related with an improvement in clinical status, exercise capacity, and quality of life. Ongoing trials are testing the hypothesis that such a therapy may also reduce the risk of HF hospitalizations and cardiovascular death.

Impact of chronic obstructive pulmonary disease on prognosis in atrial fibrillation: A report from the EURObservational Research Programme Pilot Survey on Atrial Fibrillation (EORP-AF) General Registry.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common chronic disease, being associated with both high rates of morbidity and mortality. Similarly, atrial fibrillation (AF) is associated with a higher risk of both cardiovascular (CV) events and overall mortality. The AF and COPD often coexist, but the impact of COPD on prognosis in European AF patients is unknown. METHODS: We evaluated COPD prevalence in patients enrolled in the EURObservational Research Programme Pilot Survey on Atrial Fibrillation Registry Pilot Phase. Clinical factors associated with COPD and adverse outcomes at 1-year follow-up were determined. RESULTS: In the overall cohort, a diagnosis of COPD was recorded in 339 (11.0%) of AF patients. The AF patients with COPD were more burdened with risk factors and comorbidities, including diabetes mellitus (P < .0001) and chronic heart failure (P < .0001). ß-Blockers were less likely to be prescribed to patients with COPD (P = .0007). On follow-up, AF patients with COPD had a higher risk of both CV death and all-cause death (both P < .0001), as well as for the composite outcome of any thromboembolic event/bleeding /CV death (P = .0003). Cox regression analysis found that COPD was independently associated with an increase in all-cause death (hazard ratio, 1.55; 95% CI 1.05-2.28; P = .0269). CONCLUSIONS: Chronic obstructive pulmonary disease is highly prevalent in European AF patients, and is associated with higher rates of CV death, all-cause death, and the composite outcome of any thromboembolic event/bleeding/CV death. The presence of COPD in AF patients was independently associated with all-cause death in AF patients.

Evaluation of biological activity of quantum dots in a microsystem.

The presented work aimed at systematic investigation of biological activity of CdSex S1- x /ZnS and CdSe/ZnS quantum dots (QDs), whose surface was modified with different ligands. For these studies, we used a microfluidic system combined with fluorescence microscopy techniques, which enabled analysis of cells' morphology, viability, and QDs uptake. PDMS and glass-based microfluidic system enabled the precise control of the cell environment, allowed to examine five replications of each tested QDs concentrations (statistically significant number), monitor multiple cellular events, and avoid manual preparation of QDs dilutions. We investigated the influence of the core composition and the type of surface modifiers on QDs toxicity. We also determined whether the examined nanoparticles penetrate into the cells. For all tested nanoparticles, the decrease of cells' viability was observed when increasing nanoparticles concentration. The decrease of live cells' number in microchambers and the accumulation of the nanoparticles around cultured cells were observed. The effect of hydrocarbon chain length of surface modifiers and QDs core composition on the cell viability was confirmed in our tests.

Pitfalls and capabilities of various hydrogen donors in evaluation of peroxidase-like activity of gold nanoparticles.

Catalytic nanomaterials, widely used as substitutes of peroxidase, exhibit unique properties, which are unattainable for native enzymes. However, their activity is usually examined by means of substrates developed and methods standardized for horseradish peroxidase (HRP). The aim of the presented work was to determine the scope of usefulness of chromogenic substrates for gold nanoparticle (AuNP) activity studies under conditions which significantly extend beyond the activity range of a native HRP. The applicability of chromogens such as 3,3'5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) beyond the typical range of pH, and for the samples of high concentration of hydrogen peroxide was examined. The conducted research confirmed the usefulness of ABTS and TMB in acidic media (pH 2.5-3.5). At the same time, potential interferences from chloride anion, unobservable for HRP-based assays, were indicated. Moreover, a number of potentially useful hints concerning relations of concentration of substrates and catalyst for aromatic amine oxidation (TMB and OPD) were proposed. By increasing the concentration of chromogens and thanks to assuring the relatively low conversion of the reaction, the stability of TMB and OPD oxidation product was improved even in acidic media. The comparative studies of H2O2 affinity to the surface of AuNPs in the presence of various hydrogen donors underlined the superiority of phenolic compounds over aromatic amines and ABTS in the case of the samples of relatively low H2O2 concentration. This work highlights some improvements in the methods of HRP-like activity characterization of NPs. It provides a critical analysis of the major challenges, which may emerge in a case of bioanalytical assays employing the catalytic nanoparticles as labels.

Peroxidase-like activity of gold nanoparticles stabilized by hyperbranched polyglycidol derivatives over a wide pH range.

The aim of this work was to carry out comparative studies on the peroxidase-like activity of gold nanoparticles (AuNPs) stabilized with low molecular weight hyperbranched polyglycidol (HBPG-OH) and its derivative modified with maleic acid residues (HBPG-COOH). The influence of the stabilizer to gold precursor ratio on the size and morphology of nanoparticles obtained was checked, and prepared nanoparticles were characterized by means of transmission electron microscopy and UV-Vis spectroscopy. The results indicated the divergent effect of increasing the concentration of stabilizers (HBPG-OH or HBPG-COOH) on the size of the nanostructures obtained. The gold nanoparticles obtained were characterized as having intrinsic peroxidase-like activity and the mechanism of catalysis in acidic and alkaline mediums was consistent with the standard Michaelis-Menten kinetics, revealing a strong affinity of AuNPs with 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 3, 3', 5, 5'-tetramethylbenzidine (TMB), and significantly lower affinity towards phenol. By comparing the kinetic parameters, a negligible effect of polymeric ligand charge on activity against various types of substrates (anionic or cationic) was indicated. The superiority of steric stabilization via the application of tested low-weight hyperbranched polymers over typical stabilizers in preventing salt-induced aggregation and maintaining high catalytic activity in time was proved. The applied hyperbranched stabilizers provide a good tool for manufacturing gold-based nanozymes, which are highly stable and active over a wide pH range.

A Careful Insight into DDI-Type Receptor Layers on the Way to Improvement of Click-Biology-Based Immunosensors.

Protein-based microarrays are important tools for high-throughput medical diagnostics, offering versatile platforms for multiplex immunodetection. However, challenges arise in protein microarrays due to the heterogeneous nature of proteins and, thus, differences in their immobilization conditions. This article advocates DNA-directed immobilization (DDI) as a solution, emphasizing its rapid and cost-effective fabrication of biosensing platforms. Thiolated single-stranded DNA and its analogues, such as ZNA® and PNA probes, were used to immobilize model proteins (anti-CRP antibodies and SARS-CoV nucleoprotein). The study explores factors influencing DDI-based immunosensor performance, including the purity of protein-DNA conjugates and the stability of their duplexes with DNA and analogues. It also provides insight into backfilling agent type and probe surface density. The research reveals that single-component monolayers lack protection against protein adsorption, while mixing the probes with long-chain ligands may hinder DNA-protein conjugate anchoring. Conventional DNA probes offer slightly higher surface density, while ZNA® probes exhibit better binding efficiency. Despite no enhanced stability in different ionic strength media, the cost-effectiveness of DNA probes led to their preference. The findings contribute to advancing microarray technology, paving the way for new generations of DDI-based multiplex platforms for rapid and robust diagnostics.

Sensitive and label-free SPR biosensing platforms for high-throughput screening of plasma membrane receptors interactions with insulin-like targets of hypoglycaemic activity.

Progress in medical sciences aims for tailored therapy of civilization diseases like diabetes. Preclinical screening of new medicines superior to insulin should include the verification of their affinity to the membrane receptors naturally stimulated by this hormone: insulin receptor isoforms A and B and insulin-like growth factor receptor. Considering that the affinity constants obtained using different experimental conditions are incomparable, it is essential to develop a robust and reliable method to analyze these interactions. The versatile SPR platform developed in this study enables the evaluation of the bioactivity of hypoglycaemic molecules. Thanks to the comprehensive characterization of miscellaneous aspects of the analytical platform, including the design of the SPR biosensor receptor layer, ensuring interaction specificity, as well as the quality control of the standards used (human insulin, HI; long-acting insulin analog: glargine, Gla), the feasibility of the method of equilibrium and kinetic constants determination for insulin-like targets was confirmed. SPR assays constructed in the direct format using IR-A, IR-B, and IGF1-R receptor proteins show high sensitivities and low detection limits towards insulin and glargine detection in the range of 18.3-53.3 nM with no signs of mass transport limitations. The improved analytical performance and stability of SPR biosensors favor the acquisition of good-quality kinetic data, while preservation of receptors activity after binding to long-chain carboxymethyldextran, combined with spontaneous regeneration, results in stability and long shelf life of the biosensor, which makes it useful for label-free insulin analogs biosensing and thus extensive screening in diabetic drugs discovery.

Physical Fitness Is Directly Related to Exercise Capacity and Ventilatory Response to Exercise in Men with HFrEF.

Background: Heart failure (HF) patients experience reduced functional fitness level (determining the performance of routine, daily activities) and diminished exercise capacity (linked to more effortful activities). Aim: The aim of the study is to assess this relationship using functional fitness tests compared to peak VO2 and VE/VCO2 slope in the context of exercise capacity and ventilatory response to exercise. Methods: A total of 382 men with stable HFrEF (age: 61 ± 10, NYHA class I/II/III/IV: 16/50/32/2%, LVEF: 30.5 ± 8.3%) underwent cardiopulmonary exercise testing (CPX) and a Senior Fitness Test (SFT). Afterwards, the patients were divided according to the 2capacity with peak VO2 ≥ 18 mL/kg/min, those with higher or lower ventilatory responses (VE/VCO2 slope ≥ 35 vs. <35) to the exercise were compared. Results: Patients who covered shorter distances in the 6 min walking test showed worse results in the functional tests ('stand up and go', 'chair stand' and 'arm curl') and CPX (lower peak VO2, shorter exercise time and higher VE/VCO2 slope). Subjects classified into Class D demonstrated the worst results in all elements of SFT; those in Class A demonstrated the best results. Significant differences that were analogous occurred also between classes B and C. Among the participants who reached peak VO2 ≥ 18 mL/kg/min (n = 170), those with VE/VCO2 slope ≥ 35 were characterized by worse physical fitness as compared to those with VE/VCO2 < 35. Conclusion: Reduced exercise tolerance led to worsening physical function in patients with HFrEF. Moreover, limitations in physical fitness seem to be distinctive for those patients showing excessive ventilatory response to exercise slope VE/VCO2 (≥35). The Senior Fitness Test may be considered as a useful tool for assessing comprehensive functional and clinical status and risk stratification in patients with HFrEF, especially those with extremely low exercise capacity.