Comparison of an antibody and its recombinant derivative for the detection of the small molecule explosive 2,4,6-trinitrotoluene

Antibodies are commonly used as recognition elements in immunoassays because of their high specificity and affinity, and have seen extensive use in competitive assays for the detection of small molecules. However, these complex molecules require production either in animals or by mammalian cell cultures, and are not easily tailored through genetic manipulation. Single chain antibodies (scFv), recombinantly expressed molecules consisting of only the antibody's binding region joined via a linking peptide, can provide an alternative to intact antibodies. We describe the characterization of a new monoclonal antibody (mAb), 2G5B5, able to detect the small molecule explosive 2,4,6-trinitrotoluene (TNT) and the scFv derived from its variable regions. The mAb and scFv were tested by surface plasmon resonance to determine their affinity for an immobilized TNT surrogate; dissociation constants were determined to be 1.5 × 10⁻¹³ M and 4.8 × 10⁻¹⁰ M respectively. Circular dichroism was used to determine their melting temperatures. The mAb is more stable melting at ∼75 °C while the scFv melts at ∼65 °C. The recognition elements were incorporated into a competitive assay format using a bead-based multiplexing platform to examine their sensitivity and specificity. The scFv was able to detect TNT ∼10-fold more sensitively than the mAb in this assay format, allowing detection of TNT concentrations down to at least 1 μg L⁻¹. The 2G5B gave similar detection limits to a commercial anti-TNT mAb, but was less specific, recognizing 1,3,5-trinitrobenzene (TNB) equally well as TNT.     

A reagentless DNA biosensor based on cathodic electrochemiluminescence at a C/CxO1−x electrode

A reagentless signal-on electrochemiluminescence (ECL) biosensor for DNA hybridization detection was developed based on the quenching effect of ferrocene (Fc) on intrinsic cathodic ECL at thin oxide covered glassy carbon (C/C_xO_1−x) electrodes. To construct the DNA biosensor, molecular beacon (MB) modified with ferrocene (3′-Fc) was attached to a C/C_xO_1−x electrode via the covalent bound between labeled amino (5′-NH₂) and surface functional groups. It was found that the immobilization of the probe on the electrode surface mainly depended on the fraction of surface carbonyl moiety. When a complementary target DNA (cDNA) was present, the stem-loop of MB on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the moving away of Fc from the electrode surface, and the restoring of the cathodic ECL signal. The restoration of the ECL intensity was linearly changed with the logarithm of cDNA concentration in the range of 1.0 × 10⁻¹¹ to 7.0 × 10⁻⁸ M, and the detection limit was ca. 5.0 pM (S/N = 3). Additionally, single-base mismatched DNA can be effectively discriminated from the cDNA. The great advantage of the biosensor lies in its simplicity and cost-effective with ECL generated from the electrode itself, and no adscititious luminophore is required.     

N,N′-Bis(aryl)pyridine-2,6-dicarboxamide complexes of ruthenium: Synthesis,structure and redox properties

Reaction of five N,N′-bis(aryl)pyridine-2,6-dicarboxamides (H₂L-R, where H₂ denotes the two acidic protons and R (R = OCH₃, CH₃, H, Cl and NO₂) the para substituent in the aryl fragment) with [Ru(trpy)Cl₃](trpy = 2,2′,2″-terpyridine) in refluxing ethanol in the presence of a base (NEt₃) affords a group of complexes of the type [Ru^II(trpy)(L-R)], each of which contains an amide ligand coordinated to the metal center as a dianionic tridentate N,N,N-donor along with a terpyridine ligand. Structure of the [Ru^II(trpy)(L-Cl)] complex has been determined by X-ray crystallography. All the Ru(II) complexes are diamagnetic, and show characteristic ¹H NMR signals and intense MLCT transitions in the visible region. Cyclic voltammetry on the [Ru^II(trpy)(L-R)] complexes shows a Ru(II)–Ru(III) oxidation within 0.16–0.33 V versus SCE. An oxidation of the coordinated amide ligand is also observed within 0.94–1.33 V versus SCE and a reduction of coordinated terpyridine ligand within −1.10 to −1.15 V versus SCE. Constant potential coulometric oxidation of the [Ru^II(trpy)(L-R)] complexes produces the corresponding [Ru^III(trpy)(L-R)]⁺ complexes, which have been isolated as the perchlorate salts. Structure of the [Ru^III(trpy)(L-CH₃)]ClO₄ complex has been determined by X-ray crystallography. All the Ru(III) complexes are one-electron paramagnetic, and show anisotropic ESR spectra at 77 K and intense LMCT transitions in the visible region. A weak ligand-field band has also been shown by all the [Ru^III(trpy)(L-R)]ClO₄ complexes near 1600 nm.     

Electrochemiluminescence from Ru(bpy)3 immobilized in poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate)-poly(vinyl alcohol) composite films

Electrochemical behavior and electrogenerated chemiluminescence (ECL) of tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) immobilized in poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate)-poly(vinyl alcohol) (PEDOT/PSS-PVA) composite films via ion-exchange have been investigated with tripropylamine (TPA) as the co-reactant at a glassy carbon electrode. The immobilized Ru(bpy)₃²⁺ performed a surface-controlled electrode reaction. The Ru(bpy)₃²⁺ modified electrode showed a fast ECL response to TPA, and was used for the ECL detection of TPA with high sensitivity. The ECL intensity was linearly related to concentrations of TPA over the range from 0.50 μmol L⁻¹ to 0.80 mmol L⁻¹, and the detection limit was 0.10 μmol L⁻¹ (S/N = 3). The as-prepared electrode exhibited good precision and long-term stability for TPA determination.     

Paper-based solid-state electrochemiluminescence sensor using poly(sodium 4-styrenesulfonate) functionalized graphene/nafion composite film

Herein, highly efficient solid-state ECL sensor was introduced for the first time onto the screen printed electrodes of the paper-based chips (PCs) based on the composite film of poly(sodium 4-styrenesulfonate) functionalized graphene (PSSG) and Nafion. Attributed to the cooperative characteristics of both PSS and graphene, PSSG ensured both effective Ru(bpy)₃²⁺ immobilization and fast electron transfer of Ru(bpy)₃²⁺ in the composite film. The ECL behaviors at the developed sensor were investigated using tripropylamine as a representative analyte and low detection limit (S N⁻¹ = 3) of 5.0 nM was obtained. It also exhibited more excellent reproducibility (relative standard deviations of 0.63% for continuous 45 cycles) and long-term stability (∼80% of its initial ECL intensity could be retained over 3 months). More importantly, assisted by the developed ECL sensor, discrimination of 1.0 nM single-nucleotide mismatch in human urine matrix could be realized on the PCs for the first attempt. Thus, the developed sensor was confirmed with the advantages of highly sensitivity, long-term stability, simplicity, low cost, disposability, high efficiency and potential applicability.     

3D nanostructured Ni(OH)2 microspheres as an efficient immobilization matrix of Ru(bpy)3 for high-performance electrochemiluminescence sensor

The electrochemistry and electrochemiluminescence (ECL) of novel three-dimensional nanostructured Ru(bpy)₃²⁺/Ni(OH)₂ microspheres were investigated for the first time. The negatively charged porous Ni(OH)₂ microspheres composed of Ni(OH)₂ nanowires were specifically designed to interact with Ru(bpy)₃²⁺. The large surface area and porous structure of Ni(OH)₂ microspheres enhance loading of Ru(bpy)₃²⁺ and mass transport of the model analyte, tripropylamine (TPA). Excellent ECL performance of the presented sensor was achieved including good stability and wide linear range from 7.7 × 10⁻¹⁰ to 3.8 × 10⁻³ M with the detection limit of 2.6 × 10⁻¹⁰ M to TPA.     

Electrochemiluminescence biosensor for the assay of small molecule and protein based on bifunctional aptamer and chemiluminescent functionalized gold nanoparticles

An electrochemiluminescence (ECL) biosensor for simultaneous detection of adenosine and thrombin in one sample based on bifunctional aptamer and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles (ABEI-AuNPs) was developed. A streptavidin coated gold nanoparticles modified electrode was utilized to immobilize biotinylated bifunctional aptamer (ATA), which consisted of adenosine and thrombin aptamer. The ATA performed as recognition element of capture probe. For adenosine detection, ABEI-AuNPs labeled hybridization probe with a partial complementary sequence of ATA reacted with ATA, leading to a strong ECL response of N-(aminobutyl)-N-(ethylisoluminol) enriched on ABEI-AuNPs. After recognition of adenosine, the hybridization probe was displaced by adenosine and ECL signal declined. The decrease of ECL signal was in proportion to the concentration of adenosine over the range of 5.0 × 10⁻¹²–5.0 × 10⁻⁹ M with a detection limit of 2.2 × 10⁻¹² M. For thrombin detection, thrombin was assembled on ATA modified electrode via aptamer–target recognition, another aptamer of thrombin tagged with ABEI-AuNPs was bounded to another reactive site of thrombin, producing ECL signals. The ECL intensity was linearly with the concentration of thrombin from 5 × 10⁻¹⁴ M to 5 × 10⁻¹⁰ M with a detection limit of 1.2 × 10⁻¹⁴ M. In the ECL biosensor, adenosine and thrombin can be detected when they coexisted in one sample and a multi-analytes assay was established. The sensitivity of the present biosensor is superior to most available aptasensors for adenosine and thrombin. The biosensor also showed good selectivity towards the targets. Being challenged in real plasma sample, the biosensor was confirmed to be a good prospect for multi-analytes assay of small molecules and proteins in biological samples.     

Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol

An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH = 9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H₂O₂ product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL⁻¹ to 1000 ng mL⁻¹, and a low detection limit was 0.02 ng mL⁻¹. The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing.     

The investigation of the behavior of a long period grating sensor with a copper sensitive coating fabricated by layer-by-layer electrostatic adsorption

Sensors based on changes of refractive index in response to sorption of an analyte on the coating or film of a long period grating fiber (LPG) fiber have recently been reported. In most prior work the coating or film swelled during interaction with the analyte. The swelling mechanism produced a kinetic response that slowed both the sensor's time for steady-state measurement and the reversibility of the sensor. Here, the analytical utility of fabricating these nanometer thin films using the layer-by-layer (LBL) electrostatic assembly method is evaluated using Cu^II as the test analyte and Cibacron Blue as the reagent immobilized in the LBL assembly; a generation-4 poly(amidoamine) dendrimer served as the spacer in the assembly. Detection of 1.3 mg Cu^II L⁻¹ was observed when six bilayers comprised the coating. The stable response was achieved with 0.6 mg L⁻¹ in less than 1 min. When 0.1 M HCl was used as the rinsing solution, this LPG sensor was reversible and the signal to similar concentrations of Cu^II reproducible.     

The role of direct oxalate oxidation in electrogenerated chemiluminescence of poly(4-vinylpyridine)-bound Ru(bpy)2Cl/oxalate system on indium tin oxide electrodes

The electrochemical and electrogenerated chemiluminescence (ECL) properties of indium tin oxide (ITO) electrodes modified with poly(4-vinylpyridine) (PVP)-bound Ru(bpy)₂Cl⁺ (where bpy = 2,2′-bipyridine) have been studied. In a sodium oxalate solution, two irreversible oxidation waves as well as two ECL emission waves were observed during the potential scan in the range 0.4-1.4 V (versus Ag/AgCl/saturated KCl reference). The first ECL wave appeared at ca. 0.8 V, which was caused by the excited-state Ru^2+* generated through a bimolecular redox reaction between electrogenerated Ru³⁺ and the strong reducing agent, C⁻O₂. The latter was formed via a Ru³⁺-mediated oxidation of oxalate. Direct oxidation of oxalate was not involved in the first ECL process. The second ECL wave started at ca. 1.1 V, which was also from the excited-state Ru^2+* generated via the redox reaction between Ru³⁺ and C⁻O₂. However, both direct and Ru³⁺-mediated oxidation of oxalate contributed to the formation of C⁻O₂. The important role of the direct oxidation of oxalate in the ECL mechanism of PVP-bound Ru(bpy)₂Cl⁺/oxalate system was demonstrated. The relative contribution of direct oxidation of oxalate to the observed ECL depended upon the surface concentration of PVP-bound Ru²⁺, the concentration of oxalate and the electrode potential applied.     

Mechanism study on inorganic oxidants induced inhibition of Ru(bpy)₃²+ electrochemiluminescence and its application for sensitive determination of some inorganic oxidants

Inhibited Ru(bpy)₃²⁺ electrochemiluminescence by inorganic oxidants is investigated. Results showed that a number of inorganic oxidants can quench the ECL of Ru(bpy)₃²⁺/tri-n-propylamine (TPrA) system, and the logarithm of the decrease in ECL intensity (ΔI) was proportional to the logarithm of analyte concentrations. Based on which, a sensitive approach for detection of these inorganic oxidants was established, e.g. the log-log plots of ΔI versus the concentration of MnO₄⁻, Cr₂O₇²⁻ and Fe(CN)₆³⁻ are linear in the range of 1 × 10⁻⁷ to 3 × 10⁻⁴ M for MnO₄⁻ and Cr₂O₇²⁻, and 1 × 10⁻⁷ to 1 × 10⁻⁴ M for Fe(CN)₆³⁻, with the limit of detection (LOD) of 8.0 × 10⁻⁸ M, 2 × 10⁻⁸ M, and 1 × 10⁻⁸ M, respectively. A series of experiments such as a comparison of the inhibitory effect of different compounds on Ru(bpy)₃²⁺/TPrA ECL, ECL emission spectra, UV-Vis absorption spectra etc. were investigated in order to discover how these inorganic analytes quench the ECL of Ru(bpy)₃²⁺/TPrA system. A mechanism based on consumption of TPrA intermediate (TPrA^·) by inorganic oxidants was proposed.     

Electrogenerated chemiluminescence biosensor based on Ru(bpy)3(2+) and dehydrogenase immobilized in sol-gel/chitosan/poly(sodium 4-styrene sulfonate) composite material

A new electrogenerated chemiluminescence biosensor was fabricated by immobilizing ECL reagent Ru(bpy)₃²⁺ and alcohol dehydrogenase in sol-gel/chitosan/poly(sodium 4-styrene sulfonate) (PSS) organically modified composite material. The component PSS was used to immobilize ECL reagent Ru(bpy)₃²⁺ by ion-exchange, while the addition of chitosan was to prevent the cracking of conventional sol-gel-derived glasses and provide biocompatible microenvironment for alcohol dehydrogenase. Such biosensor combined enzymatic selectivity with the sensitivity of ECL detection for quantification of enzyme substrate and it was much simpler than previous double-layer design. The detection limit was 9.3 × 10⁻⁶ M for alcohol (S/N = 3) with a linear range from 2.79 × 10⁻⁵ to 5.78 × 10⁻² M. With ECL detection, the biosensor exhibited wide linear range, high sensitivity and good stability.     

Fabrication of a novel immunosensor using functionalized self-assembled monolayer for trace level detection of TNT by surface plasmon resonance

We have developed a new immunosensor based on self-assembly chemistry for highly sensitive and label-free detection of 2,4,6-trinitrotoluene (TNT) using surface plasmon resonance (SPR). A monolayer of amine terminated poly(ethylene glycol) hydrazinehydrochloride (PEG-NH₂) thiolate was constructed on an activated gold surface and immobilized with trinitrophenyl-β-alanine (TNPh-β-alanine) by amide coupling method. The binding interaction of a monoclonal anti-TNT Ab (M-TNT Ab) with TNPh-β-alanine immobilized thiolate monolayer surface was monitored and evaluated for detection of TNT based on the principle of indirect competitive immunoreaction. Here, the competition between the self-assembled TNT derivative and the TNT in solution for binding with antibody yields in the response signal that is inversely proportional to the concentration of TNT in the linear detection range. With the present immunoassay format, TNT could be detected in the concentration range from 0.008 ng/ml (8 ppt) to 30 ng/ml (30 ppb). The response time for an immunoreaction was 2 min and one immunocycle could be done with in 4 min including surface regeneration. Bound antibodies could be easily eluted from the self-assembled immunosurface at high recoveries (more than 100 cycles) using pepsin solution without any damage to the TNT derivatives immobilized on the surface. The compact self-assembled monolayer was highly stable and prevented the non-specific adsorption of proteins on the surface favoring error free measurement.     

A novel electrochemical biosensor based on the hemin-graphene nano-sheets and gold nano-particles hybrid film for the analysis of hydrogen peroxide

Hydrogen peroxide is an important analyte in biochemical, industrial and environmental systems. Therefore, development of novel rapid and sensitive analytical methods is useful. In this work, a hemin-graphene nano-sheets (H-GNs)/gold nano-particles (AuNPs) electrochemical biosensor for the detection of hydrogen peroxide (H₂O₂) was researched and developed; it was constructed by consecutive, selective modification of the GCE electrode. Performance of the H-GNs/AuNPs/GCE was investigated by chronoamperometry, and AFM measurements suggested that the graphene flakes thickness was ∼1.3 nm and that of H-GNs was ∼1.8 nm, which ultimately indicated that each hemin layer was ∼0.25 nm. This biosensor exhibited significantly better electrocatalytic activity for the reduction of hydrogen peroxide in comparison with the simpler AuNPs/GCE and H-GNs/GCE; it also displayed a linear response for the reduction of H₂O₂ in the range of 0.3 μM to 1.8 mM with a detection limit of 0.11 μM (S N⁻¹ = 3), high sensitivity of 2774.8 μA mM⁻¹ cm⁻², and a rapid response, which reached 95% of the steady state condition within 5 s. In addition, the biosensor was unaffected by many interfering substances, and was stable over time. Thus, it was demonstrated that this biosensor was potentially suitable for H₂O₂ analysis in many types of sample.     

Tris(2,2′-bipyridyl)ruthenium(II) electrogenerated chemiluminescence sensor based on carbon nantube dispersed in sol-gel-derived titania-Nafion composite films

A highly sensitive and stable tris(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)₃²⁺) electrogenerated chemiluminescence (ECL) sensor was developed based on carbon nanotube (CNT) dispersed in mesoporous composite films of sol-gel titania and perfluorosulfonated ionomer (Nafion). Single-wall (SWCNT) and multi-wall carbon nanotubes (MWCNT) can be easily dispersed in the titania-Nafion composite solution. The hydrophobic CNT in the titania-Nafion composite films coated on a glassy carbon electrode certainly increased the amount of Ru(bpy)₃²⁺ immobilized in the ECL sensor by adsorption of Ru(bpy)₃²⁺ onto CNT surface, the electrocatalytic activity towards the oxidation of hydrophobic analytes, and the electronic conductivity of the composite films. Therefore, the present ECL sensor based on the CNT-titania-Nafion showed improved ECL sensitivity for tripropylamine (TPA) compared to the ECL sensors based on both titania-Nafion composite films without CNT and pure Nafion films. The present Ru(bpy)₃²⁺ ECL sensor based on the MWCNT-titania--Nafion composite gave a linear response (R² = 0.999) for TPA concentration from 50 nM to 1.0 mM with a remarkable detection limit (S/N = 3) of 10 nM while the ECL sensors based on titania-Nafion composite without MWCNT, pure Nafion films, and MWCNT-Nafion composite gave a detection limit of 0.1 μM, 1 μM, and 50 nM, respectively. The present ECL sensor showed outstanding long-term stability (no signal loss for 4 months).     

A sensitive electrochemiluminescence cytosensor for quantitative evaluation of epidermal growth factor receptor expressed on cell surfaces

A sensitive electrochemiluminescence (ECL) strategy for evaluating the epidermal growth factor receptor (EGFR) expression level on cell surfaces was designed by integrating the specific recognition of EGFR expressed on MCF-7 cell surfaces with an epidermal growth factor (EGF)-funtionalized CdS quantum dots (CdSQDs)-capped magnetic bead (MB) probe. The high sensitivity of ECL probe of EGF-funtionalized CdSQD-capped-MB was used for competitive recognition with EGFR expressed on cell surfaces with recombinant EGFR protein. The changes of ECL intensity depended on both the cell number and the expression level of EGFR receptor on cell surfaces. A wide linear response to cells ranging from 80 to 4 × 10⁶ cells mL⁻¹ with a detection limit of 40 cells mL⁻¹ was obtained. The EGF-cytosensor was used to evaluate EGFR expression levels on MCF-7 cells, and the average number of EGFR receptor on single MCF-7 cells was 1.35 × 10⁵ with the relative standard deviation of 4.3%. This strategy was further used for in-situ and real-time evaluating EGFR receptor expressed on cell surfaces in response to drugs stimulation at different concentration and incubation time. The proposed method provided potential applications in the detection of receptors on cancer cells and anticancer drugs screening.     

CoLn dinuclear complexes (Ln = Gd,Tb, Dy,Ho and Er) as platforms for 1,5-dicyanamide-bridged tetranuclear Co2Ln2 complexes: A magneto-structural and theoretical study

Five acetate-diphenoxo triply-bridged Co^II-Ln^III complexes (Ln^III = Gd, Tb, Dy, Ho, Er) of formula [Co(μ-L)(μ-Ac)Ln(NO₃)₂] and two diphenoxo doubly-bridged Co^II-Ln^III complexes (Ln^III = Gd, Tb) of formula [Co(H₂O)(μ-L)Ln(NO₃)₃]·S (S = H₂O or MeOH), were prepared in one pot reaction from the compartmental ligand N,N′,N′′-trimethyl-N,N′′-bis(2-hydroxy-3-methoxy-5-methylbenzyl)diethylene triamine (H₂L). The diphenoxo doubly-bridged Co^II-Ln^III complexes were used as platforms to obtain 1,5-dicyanamide-bridged tetranuclear Co^II-Ln^III complexes (Ln^III = Gd, Tb, Dy, Ho, Er). All exhibit ferromagnetic interactions between the Co^II and Ln^III ions and in the case of the Gd^III complexes, the J_CoGd were estimated to be ∼+0.7 cm⁻¹. Compound 3 exhibits slow relaxation of the magnetization.     

Valence and origin of metal-insulator transition in Mn doped SrRuO3 studied by electrical transport, X-ray photoelectron spectroscopy and LSDA+U calculation

We have studied the valence and electronic properties of Mn doped SrRuO₃ using electrical transport measurement, X-ray photoelectron spectroscopy (XPS) and local (spin) density approximation plus Coulomb interaction strength calculation (LSDA+U). The resistivity data revealed that the system undergoes transition from metal to insulator at the critical Mn doping level, x∼0.2, which is accompanied by the structural transition from orthorhombic to tetragonal crystal symmetry. Besides, the significant reduction of the spectral weight at the coherent zone (0.8 eV) of the valence band is observed for x>0.2. The core XPS spectra suggest that both the transition elements exist in the mixed ionic pair, Ru⁺⁴/Ru⁺⁵↔Mn⁺³/Mn⁺⁴. The detail analysis of the results suggests that the Coulomb correlation effect in conjugation with localization of the charge carriers predominate over the mixed ionic pair effect and responsible for the metal-insulator transition in the series.     

Ru(II) with chelating containing N4-type donor quadridentate Pd-oxime metal complexes: Syntheses, spectral characterization, thermal and catalytic properties

Five new metal complexes [Pd(LH)₂] (1), [Pd(L)₂Ru₂(bpy)₄](ClO₄)₂ (2), [Pd(L)₂Ru₂(phen)₄](ClO₄)₂ (3), [Pd(L)₂Ru₂(dafo)₄](ClO₄)₂ (4) and [Pd(L)₂Ru₂(dcbpy)₄](ClO₄)₂ (5), (where, L = ligand, bpy = 2,2′-bipyridine, phen = 1,10-phenantroline, dafo = 4,5-diazafluoren-9-one and dcbpy = 3,3′-dicarboxy-2,2′-bipyridine) have been isolated and characterized by UV-VIS, FT-IR, ¹H NMR, magnetic susceptibility measurements, elemental analysis, molar conductivity, X-ray powder techniques, thermal analyses and their morphology studied by SEM measurements. IR spectra show that the ligand acts in a tetradentate manner and coordinates N₄ donor groups of LH₂ to Pd^II ion. The disappereance of H-bonding (O−H···O) in the trinuclear Ru^II-Pd^II-Ru^II metal complexes, the Ru^II ion centered into the main oxime core by the coordination of the imino groups while the two Ru^II ions coordinate dianionic oxygen donors of the oxime groups and linked to the ligands of bpy, phen, dafo and dbpy. The X-powder results show that 1 metal complex is indicating crystalline nature, not amorphous nature. Whereas, the X-ray powder pattern of the ligand (LH₂) with 2, 3,4 and 5 exhibited only broad humps, indicating its amorphous nature. The catalytic activity of three different complexes were tested in the Suzuki coupling reaction. The 1, 4 and 5 metal complexes catalyse Suzuki coupling reaction between phenylboronic acid and arylbromides affording biphenyls. Also, the thermal results shown that the most stable complex is 1 compound while the less stable is 4 compound.