Rapid screening of multiple antibiotic residues in milk using disposable amperometric magnetosensors

Disposable amperometric magnetosensors, involving a mixture of modified-magnetic beads (MBs), for the multiplex screening of cephalosporins (CPHs), sulfonamides (SAs) and tetracyclines (TCs) antibiotic residues in milk are reported for the first time in this work. The multiplexed detection relies on the use of a mixture of target specific modified magnetic beads (MBs) and application of direct competitive assays using horseradish peroxidase (HRP)-labeled tracers. The amperometric responses measured at −0.20 V vs. the Ag pseudo-reference electrode of screen-printed carbon electrodes (SPCE) upon the addition of H₂O₂ in the presence of hydroquinone (HQ) as redox mediator, were used to monitor the extent of the different affinity reactions. The developed methodology, involving a simple and short pretreatment, allowed discrimination between no contaminated UHT and raw milk samples and samples containing antibiotic residues at the maximum residue limits (MRLs). The usefulness of the multiplexed magnetosensor was demonstrated by analyzing spiked milk samples in only 5 min. The results demonstrated that a clear discrimination of milk samples contaminated with antibiotics at their MRL level or their mixtures, allowing the identification of milk not complying with current legislation. These features make the developed methodology a promising alternative in the development of user-friendly devices for on-site analysis to ensure quality control for dairy products.     

Disposable amperometric magnetoimmunosensor for the sensitive detection of the cardiac biomarker amino-terminal pro-B-type natriuretic peptide in human serum

A novel amperometric magnetoimmunosensor using an indirect competitive format is developed for the sensitive detection of the amino-terminal pro-B-type natriuretic peptide (NT-proBNP). The immunosensor design involves the covalent immobilization of the antigen onto carboxylic-modified magnetic beads (HOOC-MBs) activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), and further incubation in a mixture solution containing variable concentrations of the antigen and a fixed concentration of an HRP-labeled detection antibody. Accordingly, the target NT-proBNP in the sample and that immobilized on the MBs compete for binding to a fixed amount of the specific HRP-labeled secondary antibody. The immunoconjugate-bearing MBs are captured by a magnet placed under the surface of a disposable gold screen-printed electrode (Au/SPE). The amperometric responses measured at –0.10 V (vs. a Ag pseudo-reference electrode), upon addition of 3,3′,5,5′-tetramethylbenzidine (TMB) as electron transfer mediator and H₂O₂ as the enzyme substrate, are used to monitor the affinity reaction. The developed magnetoimmunosensor provides attractive analytical characteristics in 10-times diluted human serum samples, exhibiting a linear range of clinical usefulness (0.12–42.9 ng mL⁻¹) and a detection limit of 0.02 ng mL⁻¹, which can be used in clinical diagnosis of chronic heart failure in the elderly and for classifying patients at risk of death after heart transplantation. The magnetoimmunosensor was successfully applied to the analysis of spiked human serum samples.     

Disposable amperometric magneto-immunosensor for direct detection of tetracyclines antibiotics residues in milk

The preparation and performance of a disposable amperometric magneto-immunosensor, involving the use of a selective capture antibody immobilized on the surface of protein G-functionalized magnetic beads (ProtG-MBs) and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of tetracyclines (TCs) residues in milk is reported. A direct competitive immunoassay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response measured at −0.2 V vs. the silver pseudo-reference electrode of the SPCE upon the addition of H₂O₂ in the presence of hydroquinone (HQ) as redox mediator was used as transduction signal. The developed methodology showed very low limits of detection (in the low ppb level) for 4 tetracycline antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. The usefulness of the magneto-immunosensor was demonstrated by analyzing UHT whole milk samples spiked with 44 ng mL⁻¹ tetracycline (TC) as well as a reference milk containing a certified oxytetracycline (OTC) content. These features, together with the short analysis time (30 min), the simplicity, and easy automation and miniaturization of the required instrumentation make the developed methodology a promising alternative in the development of devices for on-site analysis.     

Development of an Amperometric Immunosensor for the Quantification of Staphylococcus aureus Using Self‐Assembled Monolayer‐Modified Electrodes as Immobilization Platforms

Amperometric immunosensors for the detection and quantification of S. aureus using MPA self‐assembled monolayer modified electrodes for the immobilization of the immunoreagents are reported. Two different immunosensor configurations were compared. A competitive mode, in which protein A‐bearing S. aureus cells and antiRbIgG labeled with horseradish peroxidase (HRP) compete for the binding sites of RbIgG immobilized onto the 3‐mercaptopropionic acid (MPA) modified electrode, was evaluated. Moreover, a sandwich configuration in which S. aureus cells were immobilized onto the MPA SAM, and RbIgG and antiRbIgG labeled with HRP were further linked to the electrode surface, was also tested. In both cases, TTF was used as the redox mediator of the HRP reaction with H₂O₂, and it was co‐immobilized onto the MPA‐modified gold electrode. After optimization of the working variables for both configurations, the analytical performance of the amperometric measurements carried out at 0.00 V (vs. Ag/AgCl) showed that the competitive immunosensor exhibited a lower limit of detection (1.6×10⁵ S. aureus cells mL^?1), as well as a better repeatability and reproducibility of the measurements.     

Amperometric Magnetoimmunosensors for Direct Determination of D‐Dimer in Human Serum

Two different D‐dimer disposable amperometric immunosensing designs based on indirect competitive or sandwich formats and the use of carboxylic acid‐modified magnetic beads (COOH‐MBs) and screen‐printed carbon electrodes (SPCEs) have been developed and compared. In both approaches, the resulting modified MBs were magnetically captured on the surface of a SPCE which was used as the transducer for the electrochemical detection at ?0.20 V upon addition of H₂O₂, and hydroquinone (HQ). Both configurations exhibited linear ranges of clinical usefulness and detection limits quite below the clinical threshold (0.5 µg mL^?1 D‐dimer). The sandwich configuration has been successfully tested with serum samples.     

Disposable Amperometric Immunosensor for the Determination of the E‐Cadherin Tumor Suppressor Protein in Cancer Cells and Human Tissues

This paper describes the results obtained in the development of the first electrochemical immunosensor described to date for the detection of E‐cadherin (E‐cad) protein, a relevant biomarker of prognosis and metastasis in cancer, based on the use of magnetic microcarriers (MBs) and amperometric transduction at screen‐printed carbon electrodes (SPCEs). Thus, the determination of E‐cad protein involved the use of two specific antibodies against this protein (one of them labelled with HRP) in a sandwich configuration onto HOOC‐MBs. The magnetic bioconjugates were captured onto SPCEs and the amperometric transduction was performed using the H₂O₂/hydroquinone (HQ) system. Under optimal conditions, this bioplatform demonstrated a wide linear concentration range (0.50–25 ng mL^?1) and a detection limit as low as 0.16 ng mL^?1, well below the optimal cut‐off level for the E‐cad protein (defined as 10,000 ng mL^?1 for soluble E‐cad levels in serum). The developed sensor also showed a good reproducibility among measurements with seven different sensors constructed in the same manner (RSD, 5.4 %), stability for more than 15 days and good specificity towards other proteins commonly found on biological samples. The applicability of this simple handling bioplatform for the direct determination of this protein in cell lysates with different metastatic potential and extracts from paraffined‐embedded human colorectal cancer tissues of different grade were also demonstrated.     

Direct Determination of miR‐21 in Total RNA Extracted from Breast Cancer Samples Using Magnetosensing Platforms and the p19 Viral Protein as Detector Bioreceptor

A novel magnetobiosensing approach for the rapid, sensitive and selective miR‐21 detection is reported involving the use of a specific RNA probe (antimiR‐21), streptavidin‐magnetic beads (Strep‐MBs), the siRNA Binding Protein p19 as detector bioreceptor, and amperometric detection with the H₂O₂/hydroquinone (HQ) system at disposable screen‐printed carbon electrodes. The magnetosensor exhibited a dynamic range from 1.4 to 10 nM and a detection limit of 4.2 fmol of synthetic target miR‐21 without any amplification step in 75 min. The usefulness of the approach was evaluated by analyzing total RNA (RNA_t) extracted from metastatic breast cancer cell lines, human tissues and breast cytologies.     

Secondary inter­actions in gold(I) complexes with thione ligands. 5. Two ionic compounds of the form bis­(thione)gold(I) bis(4‐halobenzene­sulfon­yl)amide

Bis(1,3‐thia­zolidine‐2‐thione‐κS²)gold(I) bis­(4‐chloro­benzene­sulfonyl)amide, [Au(C₃H₅NS₂)₂](C₁₂H₈Cl₂NS₂O₄), has no imposed symmetry. Classical N—H⋯N and N—H⋯O hydrogen bonds link the residues to form chains parallel to the b axis. Weaker inter­actions involve C—H⋯O, C—H⋯Au and a number of X⋯Cl contacts (X = Cl, S or Au) clustered in the region y ≃ . In bis­(1‐methyl­imidazolidine‐2‐thione‐κS²)gold(I) bis­(4‐iodo­benzene­sulfonyl)amide, [Au(C₄H₈N₂S)₂](C₁₂H₈I₂NS₂O₄), the Au atom of the cation and the N atom of the anion lie on the twofold axis (0, y, ) in the space group C2/c. The formula unit forms a self‐contained ring with two symmetry‐equivalent N—H⋯O hydrogen bonds, and weak C—H⋯X (X = O, I or S), Au⋯I and I⋯I contacts are observed. In both compounds, the anions display extended conformations.     

Electron‐Transfer Mediator Microbiosensor Fabrication Based on Immobilizing HRP‐Labeled Au Colloids on Gold Electrode Surface by 11‐Mercaptoundecanoic Acid Monolayer

In this paper, a new strategy for constructing a mediator‐type amperometric hydrogen peroxide (H₂O₂) microbiosensor was described. An electropolymerized thionine film (PTH) was deposited directly onto a gold electrode surface. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly cross‐linked network structure. Consequently, horseradish peroxidase (HRP) was successfully immobilized on nanometer‐sized Au colloids, which were supported by thiol‐tailed groups of 11‐mercaptoundecanoic acid (11‐MUA) monolayer covalently bound onto PTH film. With the aid of the PTH mediator, HRP‐labeled Au colloids microbiosensor displayed excellent electrocatalytical response to the reduction of H₂O₂. This matrix showed a biocompatible microenvironment for retaining the native activity of the covalent HRP and a very low mass transport barrier to the substrate, which provided a fast amperometric response to H₂O₂. The proposed H₂O₂ microbiosensor exhibited linear range of 3.5 μM–0.7 mM with a detection limit of 0.05 μM (S/N=3). The response showed a Michaelis‐Menten behavior at larger H₂O₂ concentrations. The K_M^app value for the biosensors based on 24 nm Au colloids was found to be 47 μM, which demonstrated that HRP immobilized on Au colloids exhibited a high affinity to H₂O₂ with no loss of enzymatic activity. This microbiosensor possessed good analytical performance and storage stability.     

Amperometric magnetoimmunoassay for the direct detection of tumor necrosis factor alpha biomarker in human serum

An amperometric immunoassay for the determination of tumor necrosis factor alpha (TNFα) protein biomarker in human serum based on the use of magnetic microbeads (MBs) and disposable screen-printed carbon electrodes (SPCEs) has been developed. The specifically modified microbeads were magnetically captured on the working electrode surface and the amperometric responses were measured at −0.20 V (vs. Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as electron transfer mediator and H₂O₂ as the enzyme substrate. After a thorough optimization of the assay, extremely low limits of detection were achieved: 2.0 pg mL⁻¹ (36 fM) and 5.8 pg mL⁻¹ (105 fM) for standard solutions and spiked human serum, respectively. The simplicity, robustness and this clinically interesting LOD proved the developed TNFα immunoassay as a good contender for real clinical application.     

Solid polymer electrolytes based on polystyrene‐polyether block copolymers having branched ether structure

To obtain solid polymer electrolytes (SPEs) having high ionic conductivity together with mechanical integrity, we have synthesized polystyrene (PSt)‐polyether (PE) diblock copolymers via one‐pot anionic polymerization. The PSt block is expected to aggregate to act as hard fillers in the SPE to enhance the mechanical property. The PE block consists of random copolymer (P(EO‐r‐MEEGE)) of ethylene oxide (EO) and 2‐(2‐methoxyethoxy) ethyl glycidyl ether (MEEGE) in different molar ratios ([EO]/[MEEGE] = 100/0, 86/14, 75/25, 68/32, and 41/59). The introduction of the MEEGE moiety in PEO reduced the crystallinity of PEO, and the fast motion of the MEEGE side chain caused plasticization of the PE block, thereby contributing to the fast ion transport. SPEs were fabricated by mixing the obtained diblock copolymer (PSE_x) and lithium bis(trifluoromethanesulfonyl) amide (LiTFSA) with [Li]/[O] = 0.05. Ionic conductivity of the obtained SPEs was dependent on the molar ratio of EO in the PE block (x) as well as the weight fraction of PE block (f_PE) in the block copolymer. PSE_0.86 (f_PE = 0.65) exhibited high ionic conductivity (3.3 × 10^?5 S cm^?1 at 30°C; 1.1 × 10^?4 S cm^?1 at 60°C) comparable with that of P(EO‐r‐MEEGE) (PE_0.85; f_PE = 1.00) (9.8 × 10^?5 S cm^?1 at 30°C; 4.0 × 10^?4 S cm^?1 at 60°C).     

Coordination behaviour,molecular docking,density functional theory calculations and biological activity studies of some transition metal complexes of bis‐Schiff base ligand

Coordination compounds of Mn (II), Fe (III), Co (II), Ni (II), Cu (II) and Cd (II) ions were synthesized from reaction with Schiff base ligand 4,6‐bis((E)‐(2‐(pyridin‐2‐yl)ethylidene)amino)pyrimidine‐2‐thiol (HL) derived from the condensation of 4,6‐diaminopyrimidine‐2‐thiol and 2‐(pyridin‐2‐yl)acetaldehyde. Microanalytical data, magnetic susceptibility, infrared and ¹H NMR spectroscopies, mass spectrometry, molar conductance, powder X‐ray diffraction and thermal decomposition measurements were used to determine the structure of the prepared complexes. It was found that the coordination between metal ions and bis‐Schiff base ligand was in a molar ratio of 1:1, with formula [M (HL)(H₂O)₂] X_n (M = Mn (II), Co (II), Ni (II), Cu (II) and Cd (II), n = 2; Fe (III), n = 3). Diffuse reflectance spectra and magnetic susceptibility measurements suggested an octahedral geometry for the complexes. The coordination between bis‐Schiff base ligand and metal ions was through NNNN donor sites in a tetradentate manner. After preparation of the complexes, biological studies were conducted using Gram‐positive (B. subtilis and S. aureus) and Gram‐negative (E. coli and P. aeruginosa) organisms. Metal complexes and ligand displayed acceptable microbial activity against both types of bacteria.     

Magnetic beads-based bioelectrochemical immunoassay of polycyclic aromatic hydrocarbons

A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H₂O₂) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL⁻¹ was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.     

EuTZn (T=Pd, Pt, Au) with TiNiSi-type structure—Magnetic properties and Eu Mössbauer spectroscopy

The europium compounds EuTZn (T=Pd, Pt, Au) were synthesized from the elements in sealed tantalum tubes in an induction furnace. These intermetallics crystallize with the orthorhombic TiNiSi-type structure, space group Pnma. The structures were investigated by X-ray diffraction on powders and single crystals: a=732.3(2), b=448.5(2), c=787.7(2) pm, R₁/wR₂=0.0400/0.0594, 565 F² values for EuPdZn, a=727.8(3), b=443.7(1), c=781.7(3) pm, R₁/wR₂=0.0605/0.0866, 573 F² values for EuPtZn, and a=747.4(2), b=465.8(2), c=789.1(4) pm, R₁/wR₂=0.0351/0.0590, 658 F² values for EuAuZn, with 20 variables per refinement. Together the T and zinc atoms build up three-dimensional [TZn] networks with short T–Zn distances. The EuTZn compounds show Curie–Weiss behavior in the temperature range from 75 to 300 K with μ_eff=7.97(1), 7.70(1), and 7.94(1) μ_B/Eu atom and θ_P=18.6(1), 34.9(1), and 55.5(1) K for T=Pd, Pt, and Au, respectively, indicating divalent europium. Antiferromagntic ordering was detected at 15.1(3) K for EuPdZn and canted ferromagnetic ordering at 21.2(3) and 51.1(3) K for EuPtZn and EuAuZn. ¹⁵¹Eu Mössbauer spectroscopic measurements confirm the divalent nature of the europium atoms by isomer shift values ranging from −8.22(8) (EuPtZn) to −9.23(2) mm/s (EuAuZn). At 4.2 K full magnetic hyperfine field splitting is observed in all three compounds due to magnetic ordering of the europium magnetic moments.     

Radially oriented cellulose triacetate chains on gold nanoparticles

Cellulose triacetate (CTA) derivatives having a disulfide group at the reducing-end (CTA2S, CTA13S, CTA41S), with number average degrees of polymerization (DP_ns) of 2, 13 and 41, respectively, were prepared. The CTA-self-assembled gold nanoparticles (CTA2Au, CTA13Au, and CTA41Au) were obtained through the reduction of gold salt (HAuCl₄) with CTASs. The diameters (d) and the interparticle distances (L) of the gold cores were analyzed by transmission electron microscopy (TEM) observations. The d values of CTA2Au, CTA13Au, and CTA41Au, were 8.7, 7.9, and 13.4 nm respectively. The L values of CTA2Au, CTA13Au, and CTA41Au, were 2.8, 6.3, and 20.9 nm, respectively, and agreed well with the molecular length (l) of CTAS chains (ls of CTA2S, CTA13S, CTA41S = 2.0, 7.5, 21.5 nm, respectively). The hydrodynamic diameters (D) of CTAAu nanoparticles in chloroform solution, measured by dynamic light scattering (DLS), were larger than the d values and increased with the increase in the molecular length of the CTA chains. The CTAS chain was found to work as an excellent stabilizer of the gold nanoparticles in both solid state and solution. The molecular length of CTA chains controlled the size and the alignment of the gold nanoparticles. As a result, the radially oriented CTA chains on the gold nanoparticles were successfully prepared.     

Green and facile electrode modification by spark discharge: Bismuth oxide-screen printed electrodes for the screening of ultra-trace Cd(II) and Pb(II)

We report that highly effective electrode modification can be achieved by sparking process between a flat electrode substrate and a tip counter electrode. The concept is introduced by the development of Bi₂O₃-modified graphite screen printed electrodes (SPEs). SPEs were sparked with a bismuth wire at 1.2 kV under atmospheric conditions. The effect of polarity on the morphology of the sensing surface, bismuth loading and the sensitivity of the resulting sensors for the simultaneous anodic stripping voltammetric determination of Cd(II) and Pb(II) was investigated. Compared with electroplated and various bismuth precursors bulk-modified SPEs, the developed sparked electrodes exhibited considerably lower limit of detection (0.2 μg L^− 1, S/N = 3) for each target ion. Therefore, sparking technique offers a facile and green approach for the development of highly sensitive bismuth-based electrodes, and a wide-scope of applicability in the development of metal-modified sensing surfaces.     

Sensitive and selective magnetoimmunosensing platform for determination of the food allergen Ara h 1

A highly sensitive disposable amperometric immunosensor based on the use of magnetic beads (MBs) is described for determination of Ara h 1, the major peanut allergen, in only 2 h. The approach uses a sandwich configuration involving selective capture and biotinylated detector antibodies and carboxylic acid-modified MBs (HOOC-MBs). The MBs bearing the immunoconjugates are captured by a magnet placed under the surface of a disposable screen-printed carbon electrode (SPCE) and the affinity reactions are monitored amperometrically at −0.20 V (vs a Ag pseudo-reference electrode) in the presence of hydroquinone (HQ) as electron transfer mediator and upon addition of H₂O₂ as the enzyme substrate. The developed immunosensor exhibits a wide range of linearity between 20.8 and 1000.0 ng mL⁻¹ Ara h 1, a detection limit of 6.3 ng mL⁻¹, a great selectivity, a good reproducibility with a RSD of 6.3% for six different immunosensors and a useful lifetime of 25 days. The usefulness of the immunosensor was demonstrated by determining Ara h 1 in different matrices (food extracts and saliva). The results correlated properly with those provided by a commercial ELISA method offering a reliable and promising analytical screening tool in the development of user-friendly devices for on-site determination of Ara h 1.     

Multiplexed Immunosensing Platform Coupled to Hybridization Chain Reaction for Electrochemical Determination of MicroRNAs in Clinical Samples

There is an urgent need for development of rapid and inexpensive techniques for detection of microRNAs (miRNAs), which are potential biomarkers of various types of cancer. In this paper, we describe a multiplexed electrochemical platform for determination of three cancer‐relevant miRNAs: miR‐21, let‐7a and miR‐31. The strategy combines the use of magnetic beads (MBs) modified with a commercial antibody for the efficient capture of the heteroduplexes formed by hybridization of the target miRNA with DNA probe. Free non‐hybridized region of the DNA probe was thereafter hybridized with two biotin‐labeled auxiliary DNA probes in a process of hybridization chain reaction (HCR), resulting in a long hybrid bearing a large number of biotin molecules. Labeling of these multiple biotin units with streptavidin‐peroxidase conjugates allowed an amplification of the amperometric signal measured after capturing the modified MBs at a screen‐printed carbon electrode array of eight electrodes. The combined strategy demonstrated in a similar assay time significantly higher sensitivity than those previously described using modified MBs with the same capture antibody (without amplification by HCR) or a HCR strategy implemented on the surface of MBs, respectively. The methodology exhibits a good selectivity for discriminating single mismatches and was applied to the determination of the three target miRNAs in total RNA (RNA_t) extracted from various cancer cell lines and from cervical precancerous lesions.     

Batch‐injection versus Flow‐injection Analysis Using Screen‐printed Electrodes: Determination of Ciprofloxacin in Pharmaceutical Formulations

This article highlights the potential use of multi‐walled carbon‐nanotube modified screen‐printed electrodes (SPEs) for the amperometric sensing of ciprofloxacin and compares the association of batch‐injection analysis (BIA) and flow‐injection analysis (FIA) with amperometric detection. Both analytical systems provided precise (RSD<5 %) and sensitive determination of ciprofloxacin (LOD<0.1 μmol L^?1) within wide linear range (up to 200 μmol L^?1). Accuracy of both methods was attested by recovery values (93–107 %) and comparison with capillary electrophoresis. The BIA system is completely portable (especially due to association with SPEs) and provided faster analyses (130 h^?1) and more sensitive detection than the FIA system due to the higher flow rates of injection.     

XAFS investigation of [Pd(NH<Subscript>3</Subscript>)<Subscript>4</Subscript>][AuCl<Subscript>4</Subscript>]<Subscript>2</Subscript> and its thermolysis products

Thermolysis of double complex salt [Pd(NH₃)₄][AuCl₄]₂ has been studied in helium atmosphere from ambient to 350 °C. The XAFS of Pd K and Au L₃ edges and thermogravimetry measurements have been carried out to characterize the intermediates and the final product. In the temperature range 115–160 °C the complex is decomposed to form Pd(NH₃)₂Cl₂ and AuCl_4−x N _x species with x ranging from 2 to 3. Subsequent heating of the intermediate up to 300 °C leads to the total loss of NH₃. The Au–Cl and Au–Au bonds form the local environment of Au at the stage of decomposition while only four chlorine atoms are around Pd. At the temperature of 330 °C the Au and Pd nanoparticles as well as residues of palladium chloride are detected. The final product consists of separated Au and Pd nanoparticles.