Chemiluminescence Detection of Amino Acids,Peptides, and Proteins Using Tris-2,2′-Bipyridine Ruthenium (III)

Abstract

The feasibility of using the tris-2-2′-bipyridine ruthenium (III) (Ru(bpy)₃ ^{3 +}) chemiluminescent (CL) reaction for the detection of amino acids, peptides, and proteins has been studied.

Detection limits of the amino acids as determined by flow injection analysis (FIA) ranged from 20 pmol of proline to 50 nmol of asparagine. In general, amino acids containing secondary amine groups yielded the strongest responses. A reaction mechanism for Ru (bpy)₃ ^{3 +} chemiluminescence of aliphatic amines has been proposed. Studies of peptide molecules and poly-prolines showed that the peptide bond barely contributes to the detection signals. The separation of hydroxyproline and proline in synthetic collagen by HPLC with Ru (bpy)₃ ^{3 +} chemiluminescence detection has been shown to be possible.     

Comparison of homoleptic and heteroleptic 2,2′-bipyridine and 1,10-phenanthroline ruthenium complexes as chemiluminescence and electrochemiluminescence reagents in aqueous solution

We have conducted a comprehensive comparative study of Ru(bipy)₃²⁺, Ru(bipy)₂(phen)²⁺, Ru(bipy)(phen)₂²⁺, and Ru(phen)₃²⁺ as chemiluminescence and electrochemiluminescence (ECL) reagents, to address several previous conflicting observations and gain a greater insight into their potential for chemical analysis. Clear trends were observed in many of their spectroscopic and electrochemical properties, but the relative chemiluminescence or ECL intensity with a range of analytes/co-reactants is complicated by the contribution of numerous (sometimes opposing) factors. Significantly, the reversibility of cyclic voltammetric responses for the complexes decreased as the number of phenanthroline ligands was increased, due to the lower stability of their ruthenium(III) form in the aqueous solvent. This trend was also evident over a longer timescale when the ruthenium(III) form was spectrophotometrically monitored after chemical oxidation of the ruthenium(II) complexes. In general, the greater stability of Ru(bipy)₃³⁺ resulted in lower blank signals, although this effect was less pronounced with ECL, where the reagent is oxidised in the presence of the co-reactants. Nevertheless, this shows the need to compare signal-to-blank ratios or detection limits, rather than the more common comparisons of overall signal intensity for different ruthenium complexes. Furthermore, our results support previous observations that, compared to Ru(bipy)₃²⁺, Ru(phen)₃²⁺ provides greater ECL and chemiluminescence intensities with oxalate, which in some circumstances translates to superior detection limits, but they do not support the subsequent generalised notion that Ru(phen)₃²⁺ is a more sensitive reagent than Ru(bipy)₃²⁺ for all analytes.     

Immobilization of tris(1,10-phenanthroline)ruthenium with graphene oxide for electrochemiluminescent analysis

Electrochemiluminescence (ECL) of ruthenium complexes has broad applications and the immobilization of Ru(bpy)₃²⁺ has received extensive attention. In comparison with Ru(bpy)₃²⁺, Ru(phen)₃²⁺ can be immobilized more easily because of its better adsorbability. In this study, immobilization of Ru(phen)₃²⁺ for ECL analysis has been demonstrated for the first time by using graphene oxide (GO) as an immobilization matrix. The immobilization of Ru(phen)₃²⁺ is achieved easily by mixing Ru(phen)₃²⁺ with GO without using any ion exchange polymer or covalent method. The strong binding of Ru(phen)₃²⁺ with GO is attributed to both the π–π stacking interaction and the electrostatic interaction. The Ru(phen)₃²⁺/GO modified electrode was characterized by using tripropylamine (TPA) as the coreactant. The linear range of TPA is from 3 × 10⁻⁷ to 3 × 10⁻² mol L⁻¹ with the detection limit of 3 × 10⁻⁷ mol L⁻¹. The ECL sensor demonstrates outstanding long-term stability. After the storage in the ambient environment for 90 days, the ECL response remains comparable with its original signal.     

Ultrasensitive electrochemiluminescent aptasensor for ochratoxin A detection with the loop-mediated isothermal amplification

In this study, we for the first time presented an efficient, accurate, rapid, simple and ultrasensitive detection system for small molecule ochratoxin A (OTA) by using the integration of loop-mediated isothermal amplification (LAMP) technique and subsequently direct readout of LAMP amplicons with a signal-on electrochemiluminescent (ECL) system. Firstly, the dsDNA composed by OTA aptamer and its capture DNA were immobilized on the electrode. After the target recognition, the OTA aptamer bond with target OTA and subsequently left off the electrode, which effectively decreased the immobilization amount of OTA aptamer on electrode. Then, the remaining OTA aptamers on the electrode served as inner primer to initiate the LAMP reaction. Interestingly, the LAMP amplification was detected by monitoring the intercalation of DNA-binding Ru(phen)₃²⁺ ECL indictors into newly formed amplicons with a set of integrated electrodes. The ECL indictor Ru(phen)₃²⁺ binding to amplicons caused the reduction of the ECL intensity due to the slow diffusion of Ru(phen)₃²⁺–amplicons complex to the electrode surface. Therefore, the presence of more OTA was expected to lead to the release of more OTA aptamer, which meant less OTA aptamer remained on electrode for producing LAMP amplicons, resulting in less Ru(phen)₃²⁺ interlaced into the formed amplicons within a fixed Ru(phen)₃²⁺ amount with an obviously increased ECL signal input. As a result, a detection limit as low as 10 fM for OTA was achieved. The aptasensor also has good reproducibility and stability.     

Electrochemiluminescence from tris(2,2′-bipyridyl) ruthenium (Ⅱ) in the presence of aminocarboxylic acid co-reactants

Due to the highly sensitive electrochemiluminescence (ECL), tris(2,2′-bipyridyl) ruthenium(II) (Ru(bpy)32+) is often used in the field of bioarrays with the help of co-reactants. However, the generally used co-reactant, tripropylamine (TPA), is toxic, corrosive and volatile. Therefore, the search for safe, sensitive and economical co-reactants is critical. Herein, three aminocarboxylic acids, ethylenediamine-tetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and 2-hydroxyethylethylene diaminetriacetic ac...     

Characterization of Binding Properties of Cr(Phen)33+ and Ru(Phen)32+ Complexes with Human Lactoferrin

This work presents research about [Cr(phen)₃]³⁺ and [Ru(phen)₃]²⁺ interaction with human lactoferrin (HLf), a key carrier protein of ferric cations. The photochemical and photophysical properties of [Cr(phen)₃]³⁺ and [Ru(phen)₃]²⁺ have been widely studied in the last decades due to their potential use as photosensitizers in photodynamic therapy (PDT). The behavior between the complexes and the protein was studied employing UV–visible absorption, fluorescence emission and circular dichroism spectroscopic techniques. It was found that both complexes bind to HLf with a large binding constant (K_b): 9.46 × 10⁴ for the chromium complex and 4.16 × 10⁴ for the ruthenium one at 299 K. Thermodynamic parameters were obtained from the Van't Hoff equation. Analyses of entropy (ΔS), enthalpy (ΔH) and free energy changes (ΔG) indicate that these complexes bind to HLf because of entropy-driven processes and electrostatic interactions. According to circular dichroism experiments, no conformational changes have been observed in the secondary and tertiary structure of the protein in the presence of any of the studied complexes. These experimental results suggest that [Cr(phen)₃]³⁺ and [Ru(phen)₃]²⁺ bind to HLf, indicating that this protein could act as a carrier of these complexes in further applications.     

Luminescence of mixed-ligand Ru(II) chelates is there any bona fide case of dual emission?

The luminescence behavior of Ru(bpy)₂(pq)²⁺, Ru(bpy)₂(biq)²⁺, Ru(bpy)₂(NO₂-bpy)²⁺, Ru(phen)₂(pq)²⁺, Ru(phen)₂ (biq)²⁺, Ru(phen)₂ (DMCH)²⁺, Ru(bpy)₂(i-biq)²⁺ and Ru(bpy)(i-biq)₂²⁺ has been carefully studied. In no case has bona fide dual emission been observed. The reason for the previously reported dual emissions of Ru(bpy)₂(NO₂-bpy)²⁺ and Ru(phen)₂(pq)²⁺ are discussed.     

Detection of oncoprotein platelet-derived growth factor using a fluorescent signaling complex of an aptamer and TOTO

There have recently been advances in the application of aptamers, a new class of nucleic acids that bind specifically with target proteins, as protein recognition probes for biomedical study. The development of a signaling aptamer with the capability of simple and rapid real-time detection of disease-related proteins has attracted increasing interest. We have recently reported a new protein-detection strategy using a signaling aptamer based on a DNA molecular light-switching complex, [Ru(phen)₂(dppz)]²⁺. In this work we have used the commercially available DNA-intercalating dye, TOTO, to replace [Ru(phen)₂(dppz)]²⁺ for detection of oncoprotein platelet-derived growth factor BB (PDGF-BB), a potential cancer marker. Taking advantage of the high affinity of the aptamer to PDGF-BB and the sensitive fluorescence change of the aptamer–TOTO signaling complex on protein binding, PDGF-BB was detected in physiological buffer with high selectivity and sensitivity. The detection limit was 0.1 nmol L⁻¹, which was better than that of other reported aptamer-based methods for PDGF-BB, including that using [Ru(phen)₂(dppz)]²⁺. The method is very simple with no need for covalent labeling of the aptamer or probe synthesis. It facilitates wide application of the signaling mechanism to the analysis and study of cancer markers and other proteins.      

Effects of Heteropoly Acids and Surfactant on Electrochemiluminescence of Tris(2,2'-Bipyridine) Ruthenium(II)

ABSTRACT

The effects of heteropoly acids and Triton X-100 on electrochemiluminescence (ECL) of Ru(bpy)₃ ²⁺ are investigated. Triton X-100 prevents the oxidation of oxalate and results in an increase of the ECL signal. H₅SiW₁₁VO₄₀ prevents the direct oxidation of oxalate and makes the electrochemical behavior of Ru(bpy)₃ ²⁺ less reversible, which leads to a decrease of the ECL signal. In contrast, H₃PMo₁₂O₄₀ has negligible effect on ECL intensity. Some possible reasons for the effects on the ECL of Ru(bpy)₃ ²⁺ are discussed based on the adsorption of SiW₁₁VO₄₀ ^5? on electrode surface and the ion association between SiW₁₁VO₄₀ ^5? and Ru(bpy)₃ ²⁺. The signal of ECL decreases linearly with the concentration of heteropoly acid in the range from 2x10^?6 to 1x10^?4 mol 1^?1. The results indicate that ECL of Ru(bpy)₃ ²⁺ is a potential sensitive and selective detection method for heteropoly acids and hence for the elements comprised in them.     

Separation of Free Amiimo Acids on Reverse Stationary Phases Using an Alkyl Sulfonate Salt as a Mobile Phase Additive

Abstract

Alkylsulfonate (RSO₃ ^?) salts were evaluated as mobile phase additives for the separation of free amino acids on reverse stationary phases using an acidic mobile phase where the amino acids are cations. The enhanced amino acid retention is the result of two major interactions, one being retention of the RSO₃ ^? salt on the stationary phase and the other an ion exchange selectivity between the amino acid analyte cation and the RSO₃ ^? countercation, or other countercations in the mobile phase. Major mobile phase variables are: type and concentration of RSO₃ ^? salt (the studies focused on C₈SO₃ ^? salts), presence of organic modifier, type of countercation present, and mobile phase pH and ionic strength. Alkyl modified silica and polystyrenedivinyl-benzene copolymeric reverse stationary phases were compared. A mobile phase gradient, increasing per cent organic modifier was shown to be best, is necessary for separating complex mixtures of polar and nonpolar or basic amino acids. The procedure is applicable to the identification and/or determination of amino acids in mixtures or in peptides after hydrolysis.     

Determination of Hg2+ in Tap Water Based on the Electrochemiluminescence of Ru(phen)32+ and Thymine at Bare and Graphene Oxide‐Modified Glassy Carbon Electrodes

The electrochemiluminescence (ECL) of the Ru(phen)₃²⁺/thymine (T) system at bare and graphene oxide (GO)‐modified glassy carbon (GC) electrodes was utilized to determine Hg²⁺ in tap water. The ECL intensity of Ru(phen)₃²⁺ was considerably enhanced by the addition of thymine because of the occurrence of ECL reaction between them. Subsequently, the ECL intensity of Ru(phen)₃²⁺/T system rapidly decreased with the addition of Hg²⁺ because of the formation of a T‐Hg²⁺‐T complex. A linear response (R²=0.9914) was obtained over a Hg²⁺ concentration range of 1.0×10^?9 mol/L to 1.0×10^?5 mol/L with a detection limit of 3.4×10^?10 mol/L at a bare GC electrode in 0.1 mol/L phosphate buffer (pH=8.0). The detection limit can be further reduced to 4.2×10^?12 mol/L after modification of the GC electrode by GO. To verify its applicability, the proposed method was utilized to determine Hg²⁺ in tap water and simulated wastewater. The method exhibited good reproducibility and stability and thus reveals the possibility of developing a novel ECL detection method for Hg²⁺.     

Preparation and separation of mixed-ligand Fe(II) complexes containing 1,10-phenanthroline-5,6-dione as ligand

The synthesis, separation, and characterization of mixed-ligand iron(II) complexes containing 1,10-phenanthroline (phen), 1,10-phenanthroline-5,6-dione (pdon), and NCS^? are reported. The mixed-ligand complexes [Fe(phen)(pdon)₂]²⁺ and [Fe(phen)₂(pdon)]²⁺ were prepared from iron(II) sulfate hepta hydrate and both ligands. The mixture of both complexes formed regardless the ratio of the ligands or the reaction time; therefore, the complexes were separated successfully on the reversed phase (RP) Develosil RP-Aqueous [C30] 5?µm, 150?×?4.6?mm column by two different methods. The first method was the ion paired RP chromatography performed under gradient elution with acetonitrile–water containing 0.001?mol?L^?1 KPF₆ aqueous as mobile phases. The second method was the RP chromatography performed under gradient elution with methanol and water as mobile phases. The gradient elution with water–methanol as eluents was preferred for the semi preparative separations allowing one to use the complexes without further purification upon separation, different than the first method and its variations so far. Three complexes (5, 6, and 7) were characterized by electrospray ionization mass spectrometry, NMR, UV-Vis, and IR.     

Rolling cycle amplification based single-color quantum dots–ruthenium complex assembling dyads for homogeneous and highly selective detection of DNA

In this work, a new, label-free, homogeneous, highly sensitive, and selective fluorescent biosensor for DNA detection is developed by using rolling-circle amplification (RCA) based single-color quantum dots–ruthenium complex (QDs–Ru) assembling dyads. This strategy includes three steps: (1) the target DNA initiates RCA reaction and generates linear RCA products; (2) the complementary DNA hybridizes with the RCA products to form long double-strand DNA (dsDNA); (3) [Ru(phen)₂(dppx)]²⁺ (dppx = 7,8-dimethyldipyrido [3,2-a:2′,3′-c] phenanthroline) intercalates into the long dsDNA with strong fluorescence emission. Due to its strong binding propensity with the long dsDNA, [Ru(phen)₂(dppx)]²⁺ is removed from the surface of the QDs, resulting in restoring the fluorescence of the QDs, which has been quenched by [Ru(phen)₂(dppx)]²⁺ through a photoinduced electron transfer process and is overlaid with the fluorescence of dsDNA bonded Ru(II) polypyridyl complex (Ru-dsDNA). Thus, high fluorescence intensity is observed, and is related to the concentration of target. This sensor exhibits not only high sensitivity for hepatitis B virus (HBV) ssDNA with a low detection limit (0.5 pM), but also excellent selectivity in the complex matrix. Moreover, this strategy applies QDs–Ru assembling dyads to the detection of single-strand DNA (ssDNA) without any functionalization and separation techniques.     

Separation and indirect detection of alkyl sulfonates and sulfates

Iron(II) 1,10-phenanthroline, Fe(phen)3(2+), salts are used as mobile phase additives for the liquid chromatographic separation of alkyl sulfonates and sulfates on the reversed-phase PRP-1. As alkyl chain length increases retention increases. For a given chain length an alkyl sulfate is more retained than the corresponding alkyl sulfonate. Major elution variables that affect retention are mobile phase solvent and counteranion concentration. Indirect photometric detection is used to detect alkyl sulfonates and sulfates at 510 nm where Fe(phen)3(2+) salts absorb. Conditions for isocratic and gradient elution of multicomponent mixtures are described. Detection limits depending on analyte approached 0.1 nmol for isocratic elution and 3 nmol for gradient elution.     

Determination of Cu2+ in Drinking Water Based on Electrochemiluminescence of Ru(phen)32+ and Cyclam

A convenient and simple electrochemiluminescence (ECL) method was employed to detect trace amounts of Cu²⁺ in drinking water. This method is based on the inhibitory effect of Cu²⁺ on the ECL of Ru(phen)₃²⁺ and 1,4,8,11‐tetraazacyclotetradecane (cyclam) system. ECL intensity of Ru(phen)₃²⁺ was considerably enhanced by the addition of cyclam because of the ECL reaction between them. The ECL intensity of Ru(phen)₃²⁺/cyclam system rapidy decreased with the addition Cu²⁺ because of the formation of chelate complex [Cu(cyclam)]²⁺. Good linear response (R ²=0.9948) was obtained at Cu²⁺ concentration of 1.0×10⁻⁹−1.0×10⁻⁵ mol ⋅ L⁻¹ at glassy carbon electrode in 0.1 mol ⋅ L⁻¹ phosphate buffer (pH 9.0). Observed detection limit of 4.8×10⁻¹⁰ mol ⋅ L⁻¹ satisfied the maximum contaminant level goal (MCLG) for Cu²⁺ set by the US Environmental Protection Agency (US EPA). Applicability of the proposed method was verified by the good reproducibility and stability of the method when applied to determine Cu²⁺ in tap water and simulated wastewater. Thus, a novel ECL detection method was developed for Cu²⁺ detection.     

High Sensitive Electrochemiluminescence Biosensor Based on Ru(phen)32+-loaded Double Strand DNA as Signal Tags use to Detect DNA Methyltransferase Activity

DNA methyltransferase (DNA MTase) can act as biomarker for many diseases and it is important to develop some new methods for sensitive detection of DNA MTase. In this work, a highly efficient electrochemiluminescence (ECL) sensor had been designed for detection of DNA MTase based on Ru(phen)₃²⁺ loaded double strand DNA (dsDNA- Ru(phen)₃²⁺) as signal tags. Ru(phen)₃²⁺ had been efficiently embed in the dsDNA produced through a simple hybridization chain reaction. First, a hairpin probe was designed, which can be specifically recognized by Dam MTase and modified with -SH at one end. It was modified on the surface of gold electrode by -SH as an immobilization probe (IP). This IP will be methylated in the present of Dam MTase and digested by DpnI following. Results in the release of capture probe (CP) which remains on the surface of gold electrode. The CP can hybridize with the single stand part of the dsDNA- Ru(phen)₃²⁺ and make the immobilization of ECL tags on the electrode surface, which results in a strong ECL signals detected. However, without the effect of Dam MTase, the hairpin structure of IP remains stable and cannot capture signal tags, and can only detecte weak ECL signals. The biosensor can detect the activity of Dam MTase in the concentration range of 0.01 U/mL to 20 U/mL with the ECL intensity and the logarithm of the concentration have a linear relationship, and the detection limit is calculated to be 7.6 mU/mL. The developed sensor has the ability to specifically detect Dam MTase, which can be differentiated from other types of DNA MTase. In addition, the designed method has good applicability to detect Dam MTase activity in serum samples and been applied to detect its inhibitor with high efficiency.     

Evidence for symmetry reduction in excited states of [Ru(bpy)3]+ and related compounds

Photoselection and other spectroscopic data for [Ru(bpy)₃]²⁺, [Ru(phen)₃]²⁺, [Ru(bpy)(py)₄]²⁺ and [Os(bpy)₃]²⁺ suggest that the emitting state for the tris compounds may be localized on a single ring.     

Preparation of graphene platelet-Ru(phen) 3 2+ assemblies and their application in electrochemiluminescence detection

Graphene platelet (GP)-Ru(phen) ₃ ²⁺ assembles have been prepared through self-assembly of poly sodium styrenesulfonate (PSS) functionalized GPs and Ru(phen) ₃ ²⁺ driven by electrostatic attraction interactions in aqueous solution. The resultant assembled GP-Ru(phen) ₃ ²⁺ hybrid structure modified electrode exhibits excellent electrochemiluminescence (ECL) behaviors because of the ECL active species Ru(phen) ₃ ²⁺ contained therein.     

Stabilization of G‐Quadruplex DNA,Inhibition of Telomerase Activity and Live Cell Imaging Studies of Chiral Ruthenium(II) Complexes

Telomerase inhibition is an attractive strategy for cancer chemotherapy. In the current study, we have synthesized and characterized two chiral ruthenium(II) complexes, namely, Λ‐[Ru(phen)₂(p‐MOPIP)]²⁺ and Δ‐[Ru(phen)₂(p‐MOPIP)]²⁺, where phen is 1,10‐phenanthroline and p‐MOPIP is 2‐(4‐methoxyphenyl)‐imidazo[4,5f][1,10]phenanthroline. The chiral selectivity of the compounds and their ability to discriminate quadruplex DNA were investigated by using UV/Vis, fluorescence spectroscopy, circular dichroism spectroscopy, fluorescence resonance energy transfer melting assay, polymerase chain reaction stop assay and telomerase repeat amplification protocol. The results indicate that the two chiral compounds could induce and stabilize the formation of antiparallel G‐quadruplexes of telomeric DNA in the presence or absence of metal cations. We report the remarkable ability of the two complexes Λ‐[Ru(phen)₂(p‐MOPIP)]²⁺ and Δ‐[Ru(phen)₂(p‐MOPIP)]²⁺ to stabilize selectively G‐quadruplex DNA; the former is a better G‐quadruplex binder than the latter. The anticancer activities of these complexes were evaluated by using the MTT assay. Interestingly, the antiproliferative activity of Λ‐[Ru(phen)₂(p‐MOPIP)]²⁺ was higher than that of Δ‐[Ru(phen)₂(p‐MOPIP)]²⁺, and Λ‐[Ru(phen)₂(p‐MOPIP)]²⁺ showed a significant antitumor activity in HepG2 cells. The status of the nuclei in Λ/Δ‐[Ru(phen)₂(p‐MOPIP)]²⁺‐treated HepG2 cells was investigated by using real‐time living cell microscopy to determine the effects of Λ/Δ‐[Ru(phen)₂(p‐MOPIP)]²⁺ on intracellular accumulation. The results show that Λ/Δ‐[Ru(phen)₂(p‐MOPIP)]²⁺ can be taken up by HepG2 cells and can enter into the cytoplasm as well as accumulate in the nuclei; this suggests that the nuclei were the cellular targets of Λ/Δ‐[Ru(phen)₂(p‐MOPIP)]²⁺.     

Synthesis,structure and luminescence properties of two novel lanthanide complexes with 2-fluorobenzoic acid and 1,10-phenanthroline

Two lanthanide complexes with 2-fluorobenzoate (2-FBA) and 1,10-phenanthroline (phen) were synthesized and characterized by X-ray diffraction. The structure of each complex contains two non-equivalent binuclear molecules, [Ln(2-FBA)₃?·?phen?·?CH₃CH₂OH]₂ and [Ln(2-FBA)₃?·?phen]₂ (Ln?=?Eu (1) and Sm (2)). In [Ln(2-FBA)₃?·?phen?·?CH₃CH₂OH]₂, the Ln³⁺ is surrounded by eight atoms, five O atoms from five 2-FBA groups, one O atom from ethanol and two N atoms from phen ligand; 2-FBA groups coordinate Ln³⁺ with monodentate and bridging coordination modes. The polyhedron around Ln³⁺ is a distorted square-antiprism. In [Ln(2-FBA)₃?·?phen]₂, the Ln³⁺ is coordinated by nine atoms, seven O atoms from five 2-FBA groups and two N atoms of phen ligand; 2-FBA groups coordinate Ln³⁺ ion with chelating, bridging and chelating-bridging three coordination modes. The polyhedron around Ln³⁺ ion is a distorted, monocapped square-antiprism. The europium complex exhibits strong red fluorescence from ⁵D₀?→?⁷F _j ( j?=?1–4) transition emission of Eu³⁺.