Monoclonal antibody-navigated carbon-encapsulated iron nanoparticles used for MRI-based tracking integrin receptors in murine melanoma.

Integrin beta-3 is a cell adhesion molecule that mediate cell-to-cell and cell-to-extracellular matrix communication. The major goal of this study was to explore melanoma cells (B16F10) based upon specific direct targeting of the ß3 subunit (CD61) in the integrin αvß3 receptor using carbon-encapsulated iron nanoparticles decorated with monoclonal antibodies (Fe@C-CONH-anti-CD61 and Fe@C-(CH2)2-CONH-anti-CD61). Both melanoma cells treated with nanoparticles as well as C57BL/6 mice bearing syngeneic B16-F10 tumors intravenously injected with nanoparticles were tested in preclinical MRI studies. The as-synthesized carbon-encapsulated iron nanoparticles functionalized with CD61 monoclonal antibodies have been successfully used as a novel targeted contrast agent for MRI-based tracking melanoma cells expressing the ß3 subunit of the integrin αvß3 receptor.

Extracellular Vesicles as Next-Generation Diagnostics and Advanced Therapy Medicinal Products.

Extracellular vesicles (EVs) hold great promise for clinical application as new diagnostic and therapeutic modalities. This paper describes major GMP-based upstream and downstream manufacturing processes for EV large-scale production, also focusing on post-processing technologies such as surface bioengineering and uploading studies to yield novel EV-based diagnostics and advanced therapy medicinal products. This paper also focuses on the quality, safety, and efficacy issues of the bioengineered EV drug candidates before first-in-human studies. Because clinical trials involving extracellular vesicles are on the global rise, this paper encompasses different clinical studies registered on clinical-trial register platforms, with varying levels of advancement, highlighting the growing interest in EV-related clinical programs. Navigating the regulatory affairs of EVs poses real challenges, and obtaining marketing authorization for EV-based medicines remains complex due to the lack of specific regulatory guidelines for such novel products. This paper discusses the state-of-the-art regulatory knowledge to date on EV-based diagnostics and medicinal products, highlighting further research and global regulatory needs for the safe and reliable implementation of bioengineered EVs as diagnostic and therapeutic tools in clinical settings. Post-marketing pharmacovigilance for EV-based medicinal products is also presented, mainly addressing such topics as risk assessment and risk management.

Parallel SPR and QCM-D Quantitative Analysis of CD9, CD63, and CD81 Tetraspanins: A Simple and Sensitive Way to Determine the Concentration of Extracellular Vesicles Isolated from Human Lung Cancer Cells.

Tetraspanins, including CD9, CD63, and CD81, are transmembrane biomarkers that play a crucial role in regulating cancer cell proliferation, invasion, and metastasis, as well as plasma membrane dynamics and protein trafficking. In this study, we developed simple, fast, and sensitive immunosensors to determine the concentration of extracellular vesicles (EVs) isolated from human lung cancer cells using tetraspanins as biomarkers. We employed surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation (QCM-D) as detectors. The monoclonal antibodies targeting CD9, CD63, and CD81 were oriented vertically in the receptor layer using either a protein A sensor chip (SPR) or a cysteamine layer that modified the gold crystal (QCM-D) without the use of amplifiers. The SPR studies demonstrated that the interaction of EVs with antibodies could be described by the two-state reaction model. Furthermore, the EVs' affinity to monoclonal antibodies against tetraspanins decreased in the following order: CD9, CD63, and CD81, as confirmed by the QCM-D studies. The results indicated that the developed immunosensors were characterized by high stability, a wide analytical range from 6.1 × 104 particles·mL-1 to 6.1 × 107 particles·mL-1, and a low detection limit (0.6-1.8) × 104 particles·mL-1. A very good agreement between the results obtained using the SPR and QCM-D detectors and nanoparticle tracking analysis demonstrated that the developed immunosensors could be successfully applied to clinical samples.

Graphene-encapsulated iron nanoparticles as a non-viral vector for gene delivery into melanoma cells.

The rapid progress of nanotechnology has led to use different nanomaterials for biomedical applications. Among them, graphene-encapsulated magnetic nanoparticles (GEMNS) are recognized as next generation carbon nanomaterials in translation cancer research. In this study, we utilized green fluorescence protein (GFP) expression plasmid DNA (pDNA) and GEMNS decorated with branched polyethyleneimine (PEI) to yield a novel transporter (GEMNS-PEI/pDNA) for gene delivery into melanoma cells (B16F10). The efficiency of transfection was examined using PCR and confocal microscopy. The studies show that the as-designed GEMNS-PEI construct is successfully used to transfect the melanoma cells with pDNA and it should be considered as a potent non-viral vector for introducing naked nucleic acids into eucaryotic cells.

Nanocomposites as biomolecules delivery agents in nanomedicine.

Nanoparticles (NPs) are atomic clusters of crystalline or amorphous structure that possess unique physical and chemical properties associated with a size range of between 1 and 100 nm. Their nano-sized dimensions, which are in the same range as those of vital biomolecules, such as antibodies, membrane receptors, nucleic acids, and proteins, allow them to interact with different structures within living organisms. Because of these features, numerous nanoparticles are used in medicine as delivery agents for biomolecules. However, off-target drug delivery can cause serious side effects to normal tissues and organs. Considering this issue, it is essential to develop bioengineering strategies to significantly reduce systemic toxicity and improve therapeutic effect. In contrast to passive delivery, nanosystems enable to obtain enhanced therapeutic efficacy, decrease the possibility of drug resistance, and reduce side effects of "conventional" therapy in cancers. The present review provides an overview of the most recent (mostly last 3 years) achievements related to different biomolecules used to enable targeting capabilities of highly diverse nanoparticles. These include monoclonal antibodies, receptor-specific peptides or proteins, deoxyribonucleic acids, ribonucleic acids, [DNA/RNA] aptamers, and small molecules such as folates, and even vitamins or carbohydrates.

MRI-based preclinical discovery of DILI: A lesson from paracetamol-induced hepatotoxicity.

Worldwide, drug-induced liver injury (DILI) is a major cause of hepatic failure. It is also the leading cause of withdrawal, cautionary labeling, and restricted usage of licensed drugs; therefore, European Medicines Agency (EMA) and United States Food and Drug Administration (FDA) warn that the existing methods of assessing DILI are insufficient and that some of the translational biomarkers of hepatotoxicity must be relooked. Magnetic resonance imaging (MRI) seems to be a proper tool in elucidating the effects of DILI in both preclinical and clinical studies, providing excellent visualization of the morphology of the liver parenchyma. Therefore, herein, we propose preclinical MRI assessment of liver injury in experimental paracetamol-treated rats. Quantitative MRI clearly provides evidence of adverse effects in the liver tissue caused by a single overdose of paracetamol (1 g kg-1 and 1.5 g kg-1 b.w.). The results of the MRI were confirmed by the histopathological examination (H&E) of the rat liver specimen, however the adverse effects were not disclosed due to standard aminotransferase assays (ALT/AST) in rat blood serum. The results of our analysis demonstrate the successful application of MRI in the examination of paracetamol-induced hepatotoxicity in rats; it has a potential to serve as the early diagnostic tool for the prediction of DILI in preclinical evaluation.

Modulations of bovine hepatic microsomal metabolism of benzimidazoles by secondary plant metabolites.

The study was aimed to estimate the effect of plant secondary metabolites present in ruminants diet and phytogenic feed additives on liver microsomal metabolism of albendazole and fenbendazole. The selected phytocompounds comprised of flavonoids (apigenin, quercetin) and saponins (hederagenin, medicagenic acid). The experiments were performed on liver microsomal fraction obtained from routinely slaughtered cows. The intensity of albendazole and fenbendazole metabolism in the presence of flavonoids and saponins was analyzed in equimolar concentration (100 µM). The obtained results revealed that both flavonoids and saponins intensify the metabolism of albendazole and fenbendazole in bovine microsomes. In the case of albendazole, apigenin and quercetin doubled the amount of degraded drug and the amount of produced albendazole sulfoxide. Additionally, both flavonoids increased the amount of produced albendazole sulfone. Saponins, hederagenin, and medicagenic acid intensified the degradation of albendazole (1.8-fold) and the production of albendazole sulfoxide (twofold). Medicagenic acid inhibited the production of albendazole sulfone. In the case of fenbendazole, the degradation of the drug and the production of oxfendazole were increased four and five times in the presence of saponins and flavonoids, respectively. The enhancement of benzimidazoles' metabolism caused by the studied plant metabolites could change pharmacokinetics and the efficacy of benzimidazoles' treatment in cattle.

Distribution of polyethylenimine in zebrafish embryos

Background: Polyethylenimine (PEI) plays important roles in the pharmaceutical design of non-viral gene delivery systems. Due to a set of unique physicochemical properties this cationic polymer has a great potential in modern gene therapies. Objective: The aim of the present study was to determine the distribution of branched PEI (0.8 kDa) in zebrafish embryos (Danio rerio). Material and methods: Zebrafish embryos at 3 hours post-fertilization (hpf) were incubated with PEI (10 µg/ml) for 24 and 48 hours and studied using the confocal laser microscopy. Results: The obtained results show that PEI effectively distributed into the layers of the chorion and yolk sac in developing embryos due to first 24 hours of exposure. In contrast, PEI was found in the yolk, head, trunk and tail of the embryos due to prolonged treatments (48 hours). Conclusion: The study evidences a high distribution of the branched PEI (0.8 kDa) polymer in the zebrafish embryo tissues.

Development of a validated strategy for the determination of tryptamine in human cerebrospinal fluid in the presence of competitors using molecularly imprinted polymers.

This study presents a validated strategy for the determination of tryptamine in the presence of its competitors, which involves the molecularly imprinted solid-phase extraction combined with high-performance liquid chromatography coupled with fluorimetric detection. Tryptamine-imprinted microscale sorbent was produced from 4-vinylbenzoic acid and ethylene glycol dimethacrylate in methanol by precipitation polymerization, and its imprinting factor was equal to 15.4 in static experiments or 18.6 in dynamic binding experiments. The method for tryptamine determination in the presence of serotonin and l-tryptophan was validated using a complex matrix of bovine serum albumin yielding the recoveries of tryptamine that ranged between 98.7 and 107.0%. Very low limits of detection and limits of quantification for tryptamine (19.9 and 60.3 nmol/L, respectively) allow the quantification of tryptamine in human cerebrospinal fluid in the presence of tryptophan and serotonin.

Efficient strategy for the selective determination of dopamine in human urine by molecularly imprinted solid-phase extraction.

An efficient molecularly imprinted solid-phase extraction protocol was developed for the separation of dopamine (DA) from human urine. After successful validation of the analytical method using high-performance liquid chromatography coupled with fluorescence detection, a new strategy for the selective determination of DA in the presence of norepinephrine and epinephrine in human urine was presented. In the proposed protocol, the LODs and quantification for DA were 166 ± 36 and 500 ± 110 nmol/L, respectively, and the total recoveries of DA in the range of 1-15 µmol/L varied between 98.3 and 101.1%. DA was detected in the real urine samples at the level of 47-167 µg/L (0.250-0.895 µmol/L). The superiority of the novel analytical strategy was shown by comparison with the results obtained for a commercially available imprinted sorbent.

Comparison of Chemical Composition in Tuber aestivum Vittad. of Different Geographical Origin.

Truffles are prized and nutrition-rich edible hypogeous fungi. The aim of this study was a comprehensive investigation of chemical composition of Burgundy truffle (Tuber aestivum Vittad.). We tried to answer the question: what is the impact of the environment on the truffle quality. To know the nutritional value of Burgundy truffle we compared lipids, proteins, saccharides, polyphenolics, flavonoids, total sterols, ergosterol, volatile flavour and aroma compounds content in fruit bodies of the fungus collected in three different geographical regions, i.e., Poland, Slovakia, and Italy. A comparison of the above mentioned compounds is especially interesting due to environmental and climatic differences among the studied geographical regions. Results revealed that fruit bodies of T. aestivum from Poland and Slovakia possessed nearly similar content of proteins, total sterols, and saccharides. The fruiting bodies from Italy contained significantly larger amounts of most of the investigated compounds. In turn, Polish specimens had higher content of lipids and polyphenolics than Slovak and Italian ones. We have found higher similarity of volatile compounds composition between Polish and Italian specimens than those of Polish and Slovak origin.

EXCRETION OF ETHYL GLUCURONIDE IN THE URINE OF WARSAW HIGH PREFERRING RATS DEPENDS ON THE CONCENTRATION OF INGESTED ETHANOL.

Ethyl glucuronide (EtG) is a direct ethanol metabolite. The presence of EtG in urine can be used as a laboratory test to detect recent alcohol consumption. Several earlier studies in humans and in rats revealed that the same amount of ethanol ingested at different concentrations results in different blood ethanol concen- trations. The effect of different concentrations of ingested ethanol on the resulting EtG levels in urine was tested in WHP rats. The EtG concentration was also measured in rat hair. A significant (p < 0.05) decrease in the total amount of urine EtG after administration of the higher concentration (50%) ethanol solution as compared to 30% ethanol at the same dose of ethanol (3 g/kg) was observed. Median EtG concentration in rat hair of 1.5 ng/mg (range: 0.7-2.3 ng/mg) was observed. Our results demonstrate that EtG production and excretion in WHP rats is dependent on alcohol concentration administered orally. EtG levels in hair closely reflect the fate of EtG in the rat.

QUINIDINE AND DOMPERIDONE INTERACTIONS IN THE RAT EXPERIMENTAL MODEL OF REPEATED ADMINISTRATION.

This study has investigated domperidone (DOM) and quinidine (QD) interaction in the Wistar rat experimental model of repeated administration. We used nonconventional administration model consistent with occasional administration method. Difference in administration was related to sequence of domperidone alone or with quinidine dosage. Expected domperidone-quinidine interactions could have its origin both in the ability of quinidine to inhibit P-glycoprotein (P-gp) activity as well as cytochrome P450-mediated metabolism of both compounds. There also were examined kinetics of acetaminophen (PAM) administered (30 mg/kg) with domperidone as an indicator of gastric emptying, showing domperidone prokinetic activity, as well as quinidine anticholinergic activity. Domperidone (30 mg/kg) with PAM and with/without quinidine (25 mg/kg) was administered orally according to the disposition regiment different for six examined rat groups. DOM and PAM concentrations in plasma were assayed by HPLC method. Following changes were observed: domperidone did not modify the duration of the uptake phase of acetaminophen; quinidine prolongs gastric emptying time (as a result of anticholinergic action); quinidine given as the fourth or fifth dose with domperidone promotes growth of its concentration in plasma; analysis of changes in the value of AUC(0-2) at the initial three weeks of experiment suggests intensity of domperidone absorption processes, the following week increase in the value AUC(4-6) suggests inhibition of domperidone hepatic biotransformation and the mechanism of induction of absorption during domperidone administration is different from the absorption - inducing effects of quinidine. Both effects are superimposed and produce large, 2, 3-fold change in domperidone's AUC(0-6).

THE EFFECT OF METHANOL USED AS VEHICULUM ON SERUM PHENACETIN CONCENTRATION IN THE RAT.

The xenobiotic absorption process is dependent on many factors, related both to the substance and form of its administration. During administration of small amounts of drugs, the effect of vehiculum on drug fate in the body becomes also evident. The intensity of absorption depends on numerous factors not necessarily related to the substance and its formulation, and also on biotransformation and active transport processes. Additional problem is the fact that many medicines are lipophilic compounds and insoluble in the water (e.g. phenacetin). Methanol and its aqueous solutions facilitate administration to the experimental animals, in the dissolved form of a number of medicines practically insoluble in water. Taking into consideration that methanol is particularly for rats, of low toxicity, it is quite frequently applied as vehiculum. The aim of this study was to investigate the potential interactions that may occur during the use of methanol as vehiculum and compare changes when were used solution 1% of carboxymethylcellulose. The study was performed on male Wistar rats. The tests were performed using phenacetin, which is recognized as biomarker of CYP 2E 1 isoform activity. Phenacetin was given per os in a single dose of 100 mg/kg b. w. Various procedures of phenacetin administration were tested, including solubilization in methanol or suspension in 1% water solution of carboxymethylcellulose. The results of this study show that methanol influences the phenacetin bioavailability and kinetics. Comparing the administration of this drug in methanol solutions against 1% of carboxymethylcellulose, it is in the case of phenacetin triple increase in AUC0-4 h. The presence of methanol affects the shape of kinetic curves of phenacetin causing higher their course until 4 hours after administration.

Surface-Bioengineered Extracellular Vesicles Seeking Molecular Biotargets in Lung Cancer Cells.

Personalized medicine is a new approach to modern oncology. Here, to facilitate the application of extracellular vesicles (EVs) derived from lung cancer cells as potent advanced therapy medicinal products in lung cancer, the EV membrane was functionalized with a specific ligand for targeting purposes. In this role, the most effective heptapeptide in binding to lung cancer cells (PTHTRWA) was used. The functionalization process of EV surface was performed through the C- or N-terminal end of the heptapeptide. To prove the activity of the EVs functionalized with PTHTRWA, both a model of lipid membrane mimicking normal and cancerous cell membranes as well as human adenocarcinomic alveolar basal epithelial cells (A549) and human normal bronchial epithelial cells (BEAS-2B) have been exposed to these bioconstructs. Magnetic resonance imaging (MRI) showed that the as-bioengineered PTHTRWA-EVs loaded with superparamagnetic iron oxide nanoparticle (SPIO) cargos reach the growing tumor when dosed intravenously in NUDE Balb/c mice bearing A549 cancer. Molecular dynamics (MD) in silico studies elucidated a high affinity of the synthesized peptide to the α5ß1 integrin. Preclinical safety assays did not evidence any cytotoxic or genotoxic effects of the PTHTRWA-bioengineered EVs.

Effective voltammetric tool for simultaneous detection of MMP-1, MMP-2, and MMP-9; important non-small cell lung cancer biomarkers.

Simultaneous detection of multiple biomarkers can allow to reduce the costs of medical diagnostics, and thus improve the accuracy and effectiveness of disease diagnosis and prognosis. Here, for the first time, we present a low-cost, simple, and rapid method for simultaneous detection of three matrix metalloproteinases (MMP-1, MMP-2, and MMP-9) that play important roles in the progression of lung cancer. The sensor matrix was constructed using a G2 polyamidoamine dendrimer (PAMAM) containing amino, carboxyl, and sulfhydryl groups. The recognition process was based on specific enzymatic cleavage of the Gly-Ile peptide bond by MMP-1, Gly-Leu bond by MMP-2, and Gly-Met bond by MMP-9, and monitoring was done by square wave voltammetry. The activity of metalloproteinases was detected based on the change of current signals of redox receptors (dipeptides labeled with electroactive compounds) covalently anchored onto the electrode surface. The conditions of the biosensor construction, including the concentration of receptors on the sensor surface and the time of interaction of the receptor with the analyte, were carefully optimized. Under optimal conditions, the linear response of the developed method ranged from 1.0⋅10-8 to 1.0 mg⋅L-1, and the limit of detection for MMP-1, MMP-2, and MMP-9 was 0.35, 0.62, and 1.10 fg⋅mL-1, respectively. The constructed biosensor enabled us to efficiently profile the levels of active forms of MMP-1, MMP-2, and MMP-9 in tissue samples (plasma and lung and tumor extracts). Thus, the developed biosensor can aid in the early detection and diagnosis of lung cancer.

Application of biocompatible and ultrastable superparamagnetic iron(III) oxide nanoparticles doped with magnesium for efficient magnetic fluid hyperthermia in lung cancer cells.

Magnetic fluid hyperthermia (MFH) is a promising therapeutic strategy that targets malignant tissues by heating to 40-43 °C using magnetic nanoparticles (MNPs) subjected to an alternating magnetic field (AMF). In this study, novel magnetic iron(III) oxide nanoparticles doped with magnesium (Mg0.1-γ-Fe2O3(mPEG-silane)0.5) were synthesized, and their structural, chemical, and magnetic properties were analyzed using the following techniques: Fourier-transform infrared spectroscopy, Raman spectroscopy, vibrating magnetometer analysis, powder X-ray diffraction, inductively coupled plasma mass spectrometry, scanning electron microscopy, high-resolution transmission electron microscopy, and energy-dispersive X-ray spectroscopy. The as-synthesized MNPs were used as water ferrofluids for MFH under an AMF in two calorimetric setups, namely phantom and lung cancer cell (A549) models. The as-synthesized MNPs were hexagonal or rhombohedral shaped, with an average size of 27 nm. They showed a typical soft ferromagnetic behavior based on the hysteresis profile, with a magnetic saturation of 70 emu g-1 and remnant magnetization of 1.6 emu g-1. In phantom studies, the ferrofluid (3.0 mg mL-1) exposed to an AMF (18.3 kA m-1, 110.1 kHz) heated up extremely quickly, reaching more than 90 °C in the first 10 min of magnetization. In cell studies, the ferrofluid (0.25 mg mL-1) under an AMF (16.7 kA m-1, 110.1 kHz) showed a slight increase in temperature within the first 12 min, reaching a peak of ca. 43-45 °C, which was stable up to the end of the AMF exposure (45 min). Under these conditions, a pronounced cytotoxic effect on the lung cancer cells was observed (viability ca. 15-20%). No such deleterious effects were observed when the cells were treated with MNPs only without an AMF. Specific absorption rate (SAR) measurements were performed using three mathematical approaches, namely the initial slope method, the corrected slope method, and the Box-Lucas method, which ranged from ca. 429 to 596 W g-1 for phantom and cell studies. Iron(III) oxide MNPs doped with magnesium were found to be candidates for MFH in lung cancer treatments.

A rapid, selective, and ultrasensitive voltammetric and gravimetric protocol for MMP-1 active form detection.

In this paper a rapid, selective, and ultrasensitive protocol for the detection of the active form of matrix metalloproteinase-1 (MMP-1), which is a novel predictive and prognostic biomarker, was presented, which might strengthen the current predictive systems. The biosensor construction procedure was extremely simple, economical, and time-saving, as it involved only the chemisorption step of the voltammetrically active receptor (tripeptide (Cys-Gly-Ile) labeled with methylene blue (MB) and the sealing thiol. The active form of MMP-1 was recognized based on its hydrolytic activity; as a consequence, the receptor fragment (-Ile-MB) was removed from the sensor surface. The biosensors constructed were characterized by a wide dynamic concentration response range (1.0 pg mL-1-1.0 µg mL-1) and a low detection limit (33 fg mL-1), especially the biosensor with voltammetric detection, without the amplification step. One of the important advantages of the proposed biosensors is that they can be directly used to analyze the content of the active form of MMP-1 in clinical samples without the dilution step and any other preparation step.

Novel electrogravimetric biosensors for the ultrasensitive detection of plasma matrix metalloproteinase-2 considered a potential tumor biomarker.

In this study, we developed novel, simple gravimetric and voltammetric sensors for the ultrasensitive detection of active matrix metalloproteinase (MMP)-2 in plasma. The developed sensors are cost-effective, require a very less amount of reagents, and are time-saving. They detect MMP-2 based on antigen-antibody recognition and its ability to cleave glycine-leucine peptide bond. The three-dimensional bioplatform of the sensors consisted of a cationic polyethyleneimine (PEI) polymer that facilitated robust immobilization of the dipeptide labeled with anthraquinone (AQ), or antibody molecules in appropriate density, which was crucial for biosensing. Detection was performed using quartz crystal microbalance with dissipation and voltammetry. The results showed that the developed sensors were characterized by high stability, wide analytical range (2.0 pg mL-1 to 5.0 µg mL-1), and low detection limit (ca. 10 fg mL-1). They also exhibited excellent efficiency in the determination of active MMP-2 in real samples, such as blood plasma. The developed sensors may hold great promise for the early diagnosis of cancers.

Poly(amidoamine) dendrimer immunosensor for ultrasensitive gravimetric and electrochemical detection of matrix metalloproteinase-9.

Monitoring the level of matrix metalloproteinase-9 (MMP-9) and inhibiting its expression is important for the diagnosis and treatment of various diseases. However, the analysis of MMP-9 is challenging owing to its very low content in the blood, especially at the early stages of diseases. Therefore, we developed an ultrasensitive and easy-to-use immunosensor based on a three-dimensional (3D) bioplatform for the determination of the total MMP-9 concentration in plasma. The used 3D bioplatform (G2 poly(amidoamine) dendrimer; PAMAM) improved the sensitivity of the determination by significantly expanding the surface area of the receptor layer. The antigen-antibody recognition process was controlled by quartz crystal microbalance with dissipation (QCM-D) and electrochemical impedance spectroscopy (EIS). The effect of the orientation of antibody molecules in the sensing layer on the work parameters of the immunosensor was analyzed using unmodified PAMAM (PAMAM-NH2) and PAMAM functionalized with -COOH groups (PAMAM-COOH). The developed immunosensor based on PAMAM-NH2 was characterized by a lower detection limit (LOD = 2.0 pg⋅mL-1) and wider analytical range (1·10-4 - 5 µg⋅mL-1 for EIS and QCM-D) compared to PAMAM-COOH immunosensor (EIS: 1·10-4 - 0.5 µg⋅mL-1; QCM-D: 5·10-4 - 0.5 µg⋅mL-1). The functionality of the proposed device was verified in spiked plasma. The recoveries determined in commercial human and rat plasma and noncommercial rat plasma were very close to the value of 100% and in the range of 96-120% for Au/PAMAM-NH2/Ab and Au/PAMAM-COOH/Ab immunosensors, respectively. The designed analytical devices showed high selectivity and sensitivity without the use of any amplifiers such as metal nanoparticles or enzymes.