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Heterogeneous oligonucleotide-hybridization assay based on hot electron-induced electrochemiluminescence of a rhodamine label at oxide-coated aluminum and silicon electrodes
Abstract
This paper describes a heterogeneous oligonucleotide-hybridization assay based on hot electron-induced electrochemiluminescence (HECL) of a rhodamine label. Thin oxide-film coated aluminum and silicon electrodes were modified with an aminosilane layer and derivatized with short, 15-mer oligonucleotides via diisothiocyanate coupling. Target oligonucleotides were conjugated with tetramethylrhodamine (TAMRA) dye at their amino modified 5′ end and hybridization was detected using HECL of TAMRA. Preliminary results indicate sensitivity down to picomolar level and low nonspecific adsorption. The sensitivity was better on oxide-coated silicon compared to oxide-coated aluminum electrodes and two-base pair mismatched hybrids were successfully discriminated. The experimental results presented here might be useful for the design of disposable electrochemiluminescent DNA biosensors.
... The significant advantage of cathodic ECL lies in that a low detection limit and a wide linear range can be achieved at the same time [17,18] . This method has been previously used for biorecognition detection in DNA hybridization [19], however, the complicated labeling process of probe with luminophore is required. Furthermore , the oligonucleotides are physically adsorbed on the surfaces by both electrostatic and hydrophobic interactions, which would result in a poor stability and reproducibility. ...
... In order to generate energetic electrons by direct tunneling during cathodic pulse polarization, a thin oxide film with a thickness no more than 5 nm is required. In previous reports, Al/Al 2 O 3 and Si/SiO 2 electrodes are commonly used171819. A C/C x O 1−x electrode was fabricated by electrochemical oxidizing glassy carbon substrate in 0.1 M NaOH in our previous work. ...
... 1.0 nM) [41] and equivalent to that of the cathodic ECL based one (ca. 10.0 pM) [19]. The specificity of the present ECL hybridization assay was examined by detecting the ECL response of perfectly complementary targets and one-base mismatched strands at three concentrations of 0.1, 1.0 and 10.0 nM. ...
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(x)O(1-x)) electrodes. To construct the DNA biosensor, molecular beacon (MB) modified with ferrocene (3'-Fc) was attached to a C/C(x)O(1-x) electrode via the covalent bound between labeled amino (5'-NH(2)) 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 1.0x10(-11) to 7.0x10(-8)M, and the detection limit was ca. 5.0pM (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.
... Typically, Al/Al 2 O 3 and Si/SiO 2based electrode substrates are used to generate energetic electrons by direct tunneling during the cathodic pulse polarization. A nanometer scale thick oxide film (ca. 5 nm) is required (158,159,161). ...
... The most significant advantages of the cathodic ECL consist in the low detection limits and wide linear ranges achieved at the same time (158,159). Later, this method was used by Spehar-Deleze for biorecognition detection in DNA hybridization (161). Nonetheless, complicated labeling processes are still required. ...
Analytical applications of nanomaterials used in electrochemiluminescence (ECL)-based detection methods are reviewed. Among nanomaterials, carbon-based nanomaterials (carbon nanotubes, graphene), metal nanoparticles, quantum dots, inorganic metal complexes and conducting polymers are considered. The most common mechanisms of ECL detections are also described in this review. Finally, challenges and perspectives of the use of such materials in chemical analysis are discussed.
... Luminol-H 2 O 2 -based method [148,149] and hot electron-induced ECL [150] have also been reported. In the former case, immobilization of N-(4-aminobutyl)-N-etylisoluminol (ABEI), a luminol derivative, on a target ssDNA was employed for the ECL detection of the hybridization between a complementary probe ssDNA covalently linked to a polypyrrole support and the target ssDNA [149]. ...
The development of electrochemiluminescence (ECL) applications is a growing field, having the potential advantages of ECL over conventional chemiluminescence. ECL has found various applications in immunoassays, DNA probe assays, and aptasensors by employing ECL-active species as labels on biological molecules. The recent use of ECL to detect many chemically, biochemically, clinically, and environmentally important analytes is reviewed.
... [16][17][18][19][20][21][22][23][24] Many fluorophores have been applied in the biosensors development by optical transduction, such as cyanine, rhodamine, and alexa fluor, [25][26][27] however, quantum dots (QD) are promising substitutes for these fluorophores and recently have received world attention. [28][29][30][31][32][33][34][35] QD are semiconductors materials that exhibit extraordinary optical properties, such as broad absorption spectra, narrow and symmetric emission spectrum, high photostability and decay time. 36 These exceptional photophysical properties can be applied for biosensors assembly, aiming detection of human diseases, for example, botulism, hepatitis B and breast cancer. ...
This paper describes the assembly of a bioelectrode based on poly(3-aminophenol) and anti-troponin T antibody for recognition of troponin T, which is a specific biomarker for diagnosis of acute myocardial infarction. This disease causes loss of cellular components, allowing the output of molecules such as troponin T. This proteic component acts as biomarker for diagnosis of acute myocardial infarction due to their high sensitivity and specificity. Poly(3-aminophenol) was electrodeposited onto fluorine doped tin oxide (FTO) coated glass and characterized by spectroscopic methods (UV-Visible, fluorescence, infrared), electrochemical methods (cyclic voltammetry and electrochemical impedance spectroscopy) and morphological methods (laser interferometry, field emission scanning electronic microscopy, and atomic force microscopy). UV/Vis analysis indicated that poly(3-aminophenol) presents extension of conjugation, in according with fluorescence studies. Electrochemical studies indicated that poly(3-aminophenol) electrodeposited in FTO is a material with passivating characteristics for anions and capacity of retaining cationic compounds. Laser interferometry showed that poly(3-aminophenol) covers the FTO surface with a thickness off 375±75 nm. Surface images by FE-SEM and AFM have shown a full coverage on the FTO by the polymer film. The incorporation of anti-troponin T antibody on FTO electrode modified with poly(3-aminophenol) allowed effective and selective detection of cardiac biomarker troponin T, by electrochemical impedance spectroscopy (label free) and by photoluminescence, based on CdSe/ZnS quantum dots. This research shows the step by step assembly of the bioelectrode, used for detection of troponin T by impedimetric and fluorescence methods, opening the opportunity for its use in the diagnosis of others diseases.
... Generally, hot electron induced cathodic ECL is similar to common ECL apart from that light emission is initiated by hot energetic electrons from a thin insulator film-covered during cathodic pulse polarization [2]. Previous studies demonstrated that hot electron induced cathodic ECL was an effective analytical tool [3][4][5] possessing a great many advantages, such as low detection limit, wide linear range and time-resolving [6,7]. The thin insulating oxide film on the surface of the electrodes plays a key role in the cathodic ECL process [8]. ...
Hot electron induced cathodic electrochemiluminescence (ECL) was found at a disposable CdS modified screen printed carbon electrodes (CdS-SPCEs) during cathodic pulse polarization. The ECL behavior of the modified electrode was investigated with two emissions located at 520nm and 580nm. The CdS-SPCEs show very stable and reproducible ECL signal. The ECL intensity was linear with the dissolved oxygen concentration in the range of 1.7–33mg/L with a detection limit of 0.02mg/L (S/N=3). In addition, a possible mechanism for ECL behavior of the modified electrode was proposed. The developed method can be applied to detect the dissolved oxygen concentration or biochemical oxygen demand (BOD).
For hot electron-induced electrochemiluminescence (HECL), developing facile and controllable insulating layer on the electrode surface is key to the effective electron tunneling. Herein, a polystyrene modified glassy carbon electrode based the spin-coating was proposed for HECL. The constructed interface was characterized by atomic force microscopy (AFM). And HECL performance was investigated in details using the system of Ru(bpy)3²⁺ and S2O8²⁻. The as-prepared film shows the stable and reproducible HECL performance and the HECL mechanism was discussed using ECL spectra and hydrated electron quenchers. Finally, on the basis of the inhibition of SO4•-, the present polystyrene film was applied to the detection of rutin in the range of 0.1-1.3 μM with a detection limit of 90 nM (S/N = 3).
The research progress of rhodamine fluorescent dyes was described. The structure characteristics and syntheses of them were introduced according to the modification of different position of the rhodamine derivatives. The strategies for synthesis of rhodamine derivatives and the relationship between the structure and function of them were summarized, and the significance of their applications in the field of biomolecular labeling and environmental analysis was highlighted.
In this chapter, we discuss the basics of cathodic hot electron-induced electrogenerated chemiluminescence (HECL). In the
applications of HECL, we discuss, e.g., the usable electrode materials and their advantages as well as the applicable solution
conditions in aqueous media. We also summarize the luminophore types excitable by this method and their usability as labels
in practical bioaffinity assay applications.
Electrogenerated luminescence of lanthanides is reviewed with emphasis on the electrochemiluminescence (ECL) of lanthanide chelates. Main application area of lanthanide chelates in this field is their use as electrochemiluminescent labels in bioaffinity assays such as in immunoassays or DNA probe assays. With lanthanide chelates as labels, hot electron-induced ECL at thin insulating film- coated cathodes outperforms ECL based on traditional electrochemistry at active metal electrodes. ECL excitation of lanthanide(III) chelates occurs normally by ligand-sensitized mechanisms wherein the ligand is first excited by redox reactions followed by energy transfer from ligand to the central ion, which finally emits by f–f transitions. Luminescent lanthanide ions can be excited at oxide-coated metal electrodes when these ions act as luminescence centers in the oxide film and/or at the oxide/electrolyte interface or as solvated in the close vicinity of the electrode surface. These ions typically show high field-induced solid-state electroluminescence when embedded inside of the oxide films and ECL at the surface of the electrode or in solution close to the electrode surface. These lanthanide-doped oxide films can be fabricated either by anodization of certain lanthanide-doped valve metals or from pure valve metals by anodization in lanthanide ion-containing anodization bath preferably with AC voltages. Some lanthanide ions can be electrically excited also in strongly acidic sulfuric acid solutions at platinum electrodes with mechanisms not known with certainty.
The fundamentals of electrogenerated chemiluminescence (ECL), its unique features as compared to other luminescence, and mechanisms of its several types have been covered including a review of new developments in the field of instrumentation and luminophores and applications with emphasis on the detection and determination of biorelated species. ECL is proven to be a powerful tool for ultrasensitive biomolecule detection and quantification. The miniaturized biosensors based on this technology is capable of multiplexing detection with high sensitivity, low detection limit, and good selectivity and stability.
Thin antimony oxide covered AuSb alloy electrode was firstly found to be an excellent cold cathode for generating hot electrons during cathodic pulse polarization. Owing to the injection of hot electrons and the subsequent generation of hydrated electrons, fluorescein iso-thiocyanate (FITC) that cannot be excited in common ECL was cathodically excited at the alloy electrode. Self-assembled thiol monolayers were formed on the electrode surface due to the presence of Au in the alloy, to which strepavidin was covalently bound, and then biotinylated antibody was immobilized through the strepavidin-biotin interaction. As a simple model, an immunosensor for the detection of human IgG (hIgG) using FITC as labeling agent was fabricated. ECL signals were responsive to the amount of hIgG bounded to the immunosensor. The ECL intensity was linearly changed with the logarithm of hIgG concentration in the range of 1.0-1000 ng mL(-1), and the detection limit was ca. 0.3 ng mL(-1) (S/N=3). The proposed immunosensor showed a broad linear range (three magnitudes), good reproducibility and stability, which is promising in detecting FITC-based labels in various types of bioaffinity assays.
Cathodic electrochemiluminescence (ECL) is firstly observed at a carbon oxide covered glassy carbon (C/C(x)O(1-x)) electrode as a large cathodic pulse polarization is applied. This insulating carbon oxide (C(x)O(1-x)) film is constructed on a glassy carbon (GC) substrate by electrochemical oxidization in basic media. The film properties, such as the composition of carbon and oxygen, and the thickness as well, can be controlled by the potential and the duration in the oxidizing process. X-Ray photoelectron spectroscopy (XPS) studies show that carbonyl and carboxyl dominate at the oxidized surface, to which antibodies can be covalently bound. The specific immunoreaction between antigen (Ag) and antibody (Ab) resulted in a decrease in the ECL intensity, thus creating an interesting basis for the development of a label-free cathodic ECL immunosensor. As an example, human IgG (hIgG) was sensitively determined in the concentration range of 0.01-100 ng mL(-1), and the detection limit was ca. 1.0 pg mL (-1) (S/N = 3). In addition, the content of hIgG in human serum has been assayed by the developed immunosensor and a commercially available immune turbidimetry method, respectively, and consistent results were obtained. The prepared immunosensor provides a promising approach for the clinical determination of IgG levels in human serum, because it is simple, rapid, highly sensitive, specific, and without the need of tedious labeling operations.
We herein report a DNA sensor for the sequence-specific DNA detection by using glucose oxidase-based biocatalyzed electrochemiluminescence and non-fouling surfaces. In this design, a glucose oxidase-labeled sandwich-type DNA sensor was built on a non-fouling surfaces made of a mixed self-assembled monolayers (SAMs) incorporating thiolated oligonucleotides and oligo(ethylene glycol) (OEG) thiols (SH-DNA/OEG). The sequence-specific DNA sensing was accomplished by the electrochemiluminesce (ECL) signal of luminol with the in-situ generated H(2)O(2). The protein-resistant non-fouling surfaces significantly suppressed the non-specific adsorption of enzyme label on electrode and reduced the background noise of this sensor. This sensor was able to detect as little as 1 pM of target DNA in pure buffer matrix. In complicated biological fluids such as human serum, this non-fouling platform-based sensor also revealed superior performance over conventional sandwich-type DNA sensors with mercaptohexanol (MCH)-coated surfaces. This strategy combines the high sensitivity of enzymatic amplified ECL and protein-resistant non-fouling platform, which is expected to be extended to a range of biological detection.
Homo-oligomer DNA strands were immobilized onto silicon/silicon dioxide electrodes using 3-aminopropyltriethoxysilane. These modified substrates were used as working electrodes in a three-electrode electrochemical cell. In-phase and out-of-phase impedances were measured in the range −1 to +1 V with respect to an Ag/AgCl reference electrode, with a superimposed 10 mV ac signal at frequencies of 20 and 100 kHz. Ex situ hybridization with complementary oligomer strands, performed at the surface of modified electrodes, is clearly reflected by negative shifts of about 100 mV in the flat-band potential of the semiconductor. Consecutive hybridization−denaturation steps show that the shifts are reproducible and the process is reversible. The in situ hybridization of complementary strands has also been observed with impedance measurements at Si/SiO2 substrates and with the use of a field effect device. The direct detection of hybridization with a field effect device was performed under constant drain current mode, and the corresponding variations observed for the gate potential during hybridization are in good agreement with the flat band potential shifts observed with the impedance experiments. Measurements made in the presence of noncomplementary strands demonstrate the selectivity of the device.
Electrochemiluminescence (ECL) has been developed as a highly sensitive process in which reactive species are generated from stable precursors (i.e., the ECL-active label) at the surface of an electrode. This new technology has many distinct advantages over other detection systems: no radioisotopes are used; detection limits for label are extremely low (200 fmol/L); the dynamic range for label quantification extends over six orders of magnitude; the labels are extremely stable compared with those of most other chemiluminescent systems; the labels, small molecules (approximately 1000 Da), can be used to label haptens or large molecules, and multiple labels can be coupled to proteins or oligonucleotides without affecting immunoreactivity, solubility, or ability to hybridize; because the chemiluminescence is initiated electrochemically, selectivity of bound and unbound fractions can be based on the ability of labeled species to access the electrode surface, so that both separation and nonseparation assays can be set up; and measurement is simple and rapid, requiring only a few seconds. We illustrate ECL in nonseparation immunoassays for digoxin and thyrotropin and in separation immunoassays for carcinoembryonic antigen and alpha-fetoprotein. The application of ECL for detection of polymerase chain reaction products is described and exemplified by quantifying the HIV1 gag gene.
The covalent attachment of thiol-modified DNA oligomers to self-assembled monolayer silane films on fused silica and oxidized
silicon substrates is described. A heterobifunctional crosslinking molecule bearing both thiol- and amino-reactive moieties
was used to tether a DNA oligomer (modified at its terminus with a thiol group) to an aminosilane film formed on silica surfaces.
A variety of aminosilanes, crosslinkers and treatment conditions have been tested to identify optimal conditions for DNA immobilization
using this approach. The DNA films which result have been characterized using UV spectroscopy, water contact angle measurement,
radiolabeling and hybridization methods.
Ruthenium(II) tris-(2,2′-bipyridine) chelate exhibits strong electrogenerated chemiluminescence during cathodic pulse polarization of oxide-covered aluminum electrodes in aqueous solutions. The present method is based on a tunnel emission of hot electrons into an aqueous electrolyte solution. The method allows the detection of ruthenium(II) tris-(2,2′-bipyridine) and its derivatives below nanomolar concentration levels and yields linear log-log calibration plots spanning several orders of magnitude of concentration. This method allows simultaneous excitation of derivatives of ruthenium(II) tris-(2,2′-bipyridine) and Tb(III)-chelates. The former label compounds have a luminescence lifetime of the order of microseconds, while the latter compounds generally have a luminescence lifetime of around 2 ms. Thus, the combined use of these labels easily provides the basis for two-parameter bioaffinity assays by either using wavelength or time discrimination or their combination.
An immunoanalytical method based on electrogenerated luminescence was developed in which the labelling compound is the terbium chelate of 2,6-bis[N,N-bis(carboxymethyl)aminomethyl]-4-benzoylphenol which is linked by a thioureido group to the antibody. The sandwich complex of the labelled antibody, antigen and antibody immobilized on the surface of an aluminium electrode is excited by an electrical pulse and, after a short delay from the end of the pulse, light emission is measured. The electroluminescence immunoassay is exemplified by the heterogeneous and homogeneous assays of human pancreatic phospholipase A2, for which both assay methods give a reasonably good linearity in the range 10–300 ng ml−1.
A versatile chemistry utilizing the homobifunctional cross-linker 1,4-phenylene diisothiocyanate (PDC) to attach both amine- and thiol-terminated oligonucleotides to aminosilane-coated slides was examined in a microarray format. Three common aminosilanes, 3-aminopropyltriethoxysilane (APS), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane, and m,p-(aminoethyl-aminomethyl) phenethyltrimethoxysilane, were coated onto glass slides and silicon wafers and characterized using contact angle goniometry, ellipsometry, and X-ray photoelectron spectroscopy. Evaluation of the aminosilane-modified surfaces using contact angle measurements, UV−vis spectroscopy, and covalent attachment of a Cy5-conjugated N-hydroxysuccinimide ester reporter molecule suggested that derivatization of the surface with APS + PDC resulted in the best overall coverage. Microarrays printed using APS + PDC chemistry to immobilize both amine- and thiol-terminated oligonucleotides resulted in rapid attachment, uniform spot morphology, and minimal background fluorescence. Both amine- and thiol-terminated oligonucleotides showed comparable attachment, although greater attachment and hybridization efficiencies were observed with amine-functionalized molecules at saturating printing densities. The data highlight the influence of surface chemistry on both immobilization and hybridization and, by extrapolation, on microarray data analysis.
Four kinds of aminosilane layers on glass slides or silicon wafers were prepared. The amine densities of the layers prepared with aminopropyldiethoxymethylsilane (APDES), aminopropylmonoethoxydimethylsilane (APMES), a mixture of (aminopropyl)triethoxysilane (APTES) and n-butyltrimethoxysilane (n-BTMS) (v/v = 1:10) are 4.0(±0.8), 1.0(±0.2), and 0.30(±0.6) amine/nm2, respectively. A substrate with much higher amine density, that is, 40(±8) amines/nm2 was also prepared by allowing aziridine to polymerize on the APDES-treated substrate. AFM revealed that APDES-, APMES-, and APTES/n-BTMS-treated surfaces were relatively flat; on the other hand, an aziridine-treated surface showed embossed morphology. The amine substrates were allowed to react with a heterobifunctional linker succinimidyl 4-maleimido butyrate (SMB), and subsequently pentadecadeoxynucleotides were microarrayed on the SMB-treated substrates. Characteristics of the DNA microarrays including the dynamic range, the mismatch discrimination efficiency, and so forth were examined. Noteworthily, DNA microarrays on the aziridine-polymerized substrate showed much higher fluorescence intensity. At the same time, DNA microarrays from these four substrates were able to discriminate internal- and terminal-mismatched pairs, but the fluorescence ratio was far from the one that thermodynamics implies.
The formation of thin films and monolayers of (3-aminopropyl)triethoxysilane (APS) on aluminum and gold substrates was studied with reflection-absorption FTIR spectroscopy (RAIR), ellipsometry, contact-angle, and quartz-crystal microbalance (QCM) measurements. The structure of the APS films is strongly dependent on the experimental conditions, such as substrate pretreatment, mode of adsorption, and posttreatment. APS was adsorbed on the substrates from aqueous solution, from refluxing solvent, and from the gas phase. Adsorption from solution yields multilayers. APS films of about molecular thickness are obtained by vapor adsorption on the precleaned substrate. The presence of water has a strong effect on the film formation. Depending on the experimental conditions, the final surface loading varies between about 5.3 and 8 molecules per nm(2), and the film thickness varies between 5 and 11 Angstrom.
Ruthenium(II) tris-(2,2‘-bipyridine) chelate exhibits strong electrogenerated chemiluminescence during cathodic pulse polarization of oxide-covered aluminum electrodes in aqueous solutions. The present method is based on a tunnel emission of hot electrons into an aqueous electrolyte solution. The method allows the detection of ruthenium(II) tris-(2,2‘-bipyridine) and its derivatives below nanomolar concentration levels and yields linear log−log calibration plots spanning several orders of magnitude of concentration. This method allows simultaneous excitation of derivatives of ruthenium(II) tris-(2,2‘-bipyridine) and Tb(III)-chelates. The former label compounds have a luminescence lifetime of the order of microseconds, while the latter compounds generally have a luminescence lifetime of around 2 ms. Thus, the combined use of these labels easily provides the basis for two-parameter bioaffinity assays by either using wavelength or time discrimination or their combination.
Luminol exhibits strong electrogenerated chemiluminescence during cathodic pulse polarization of oxide-covered aluminum electrodes in aqueous solution. This electrogenerated chemiluminescence can be enhanced by the presence of dissolved oxygen or by the addition of other coreactants such as hydrogen peroxide, peroxydisulfate, or peroxydiphosphate ions. However, luminol detection is most sensitive in the presence of azide ions, which not only enhance the electrogenerated chemiluminescence intensity but also decrease the intrinsic electroluminescence of the thin aluminum oxide film on the electrodes mainly producing the blank emission. The present method is based on tunnel emission of hot electrons into an aqueous electrolyte solution and allows the detection of luminol, isoluminol, and its derivatives below nanomolar concentration levels. The linear logarithmic calibration range covers several orders of magnitude of concentration of luminol or N-(6-aminohexyl)-N-ethylisoluminol. Therefore, the above-mentioned labeling substances can be used as one of several available alternatives of simultaneous markers in multiparameter bioaffinity assays at disposable oxide-covered aluminum electrodes. The main advantage of the present electrochemiluminescence generation method is that luminescent compounds having very different photophysics and chemistry can be simultaneously excited, thus providing good possibilities for internal standardization and multiparameter bioaffinity assays.
Cathodic pulse polarisation of oxide-covered aluminum electrodes in Tl(I) solutions induce strong Tl(I)-specific electrogenerated luminescence (EL). It is mainly regarded as electrochemiluminescence, induced by one-electron oxidation of cathodically produced thallium atoms in the close vicinity of the electrode surface, but solid state electroluminescence is also produced, especially, with thicker oxide films. Depending on the EL system applied, the oxidant in response of the excitation event is either a cathodically produced hydroxyl or sulfate radical, or an F+-centre of the thin oxide film, and the source of short-lived thallium atom colloids is a reduction of Tl(I) ions by tunnel-emitted hot electrons and/or cathodically generated hydrated electrons. The present method allows the detection of Tl(I) ions below nanomolar concentration level and provides linear log-log calibration graphs spanning several orders of magnitude of concentration of Tl(I).
Hot electrons can be injected from conductor/insulator/electrolyte (C/I/E) junctions into an aqueous electrolyte solution by cathodic pulse-polarization of the electrode. Injected hot electrons induce electrogenerated chemiluminescence of various luminophores including coumarins in fully aqueous solutions. This is based on the tunnel emission of hot electrons into aqueous electrolyte solution, which can result in the generation of hydrated electrons as reducing mediators. These tunnel-emitted electrons allow also the production of highly oxidizing radicals from added precursors. This work shows that coumarin derivatives are suitable candidates as ECL labels for bioaffinity assays or other analytical applications in which detection is based on the ECL of pulse-polarized C/I/E tunnel-emission electrodes in fully aqueous solutions. The mechanisms of the ECL of coumarins are discussed and the analytical applicability of the ECL of three coumarin derivatives is studied.
Aromatic Gd(III) and Y(III) chelates produce ligand-centered emissions during cathodic pulse polarization of oxide-covered
aluminum electrodes, while the corresponding Tb(III) chelates produce metal-centered5D4 →7Fj emissions. It was observed that a redox-inert paramagnetic heavy lanthanoid ion, Gd(III), seems to enhance strongly intersystem
crossing in the excited ligand and direct the deexcitation toward a triplet-state emission, while a lighter diamagnetic Y(III)
ion directs the photophysical processes toward a singlet-state emission of the ligand. The luminescence lifetime of Y(III)
chelates was too short to be measured with our apparatus, but the luminescence lifetime of Gd(III) chelates was between 20
and 70 μs. The mechanisms of the ECL processes are discussed in detail.
High amplitude cathodic pulse polarization of ultra thin oxide film-coated heavily doped silicon electrodes induces tunnel emission of hot electrons into aqueous electrolyte solution, which probably results in the generation of hydrated electrons in the vicinity of the electrode surface. The method allows the detection of tris (2,2'-bipyridine) ruthenium(II) chelate at subnanomolar concentration level. This paper shows that both n- and p-type heavily doped silicon electrodes can be used, illustrates the effect of oxide film thickness upon the silicon electrode on the intensity of ECL of tris (2,2'-bipyridine) ruthenium(II) and discusses the basic features of tris (2,2'-bipyridine) ruthenium(II) chelate-specific ECL at these electrodes. Thin oxide film-coated silicon electrodes provide a lower blank emission and a higher ECL intensity of the present ruthenium chelate than oxide-covered aluminium electrodes. This suggests that thin oxide film-coated silicon is a very promising working electrode material, especially in microanalytical systems made fully or partly of silicon. (c) 2004 Elsevier B.V. All rights reserved.
An electrochemical DNA hybridization biosensor which exploits long-range electron transfer through double-stranded DNA (ds-DNA) to a redox intercalator is described. The DNA recognition interface consisted of a mixed self-assembled monolayer of synthetic thiolated single-stranded DNA (ss-DNA) and 6-mercapto-1-hexanol (MCH). The target DNA detection is performed electrochemically through cyclic and Osteryoung square wave voltammetry, using anthraquinone derivatives as the intercalators. This biosensor has the ability to differentiate complementary target ss-DNA from non-complementary target, and most importantly, it is able to detect single-base mismatch target ss-DNA through diminution in voltammetric current. The viability of this biosensor has also been investigated through selectivity studies in the presence of interferences and the generality of the detection scheme.
Heterogeneous and homogeneous immunoassays of human thyroid stimulating hormone (hTSH) were developed on immunometric basis using aromatic Tb(III) chelates as electrochemiluminescent labels and varied types of disposable oxide-covered aluminum electrodes as the solid phase of the immunoassays. The long luminescence lifetime of the present labels allows the use of time-resolved electrochemiluminescence detection and provide the low detection limits of these labels and, thus, sensitive immunoassays. The primary antibody of immunometric immunoassays was coated upon aluminum oxide surface by physical absorption. In homogeneous immunoassays using 66 μl cell and 15 min incubation time, a linear calibration range of 0.25–324 μU/ml was obtained by applying only a single cathodic excitation pulse in the detection step of the assay.
The detection and quantification of DNA strands using an electrochemiluminescence (ECL) instrument with a microfabricated excitation/detection cell is presented. The microcell is a vertical assembly of silicon and glass substrates, containing thin film Pt electrodes and micromachined fluid channels. A silicon PIN diode photodetector completes the cell assembly. The DNA strands are labeled with an electrochemically exited, luminescing molecule: tris (2,2′ bipyridyl) ruthenium (TBR). The labeled DNA strands are bound to paramagnetic beads with a biotin–streptavidin linkage. The magnetic beads are attracted to the Pt electrode surface with an external magnet, where ECL is excited. With a 1 cm2, Pt anode, a detection limit of 40 fmol of DNA is obtained with a silicon PIN diode photodetector operating at room temperature. The overall size of the microcell is only 1.5 cm×2 cm×0.2 cm and supports a sample volume of 150 μl. This is the first demonstration of DNA quantification using an ECL microinstrument.
Oxide-covered aluminium electrodes as well as other tunnel emission electrodes allow various label molecules having very different redox and optical properties to be excited cathodically. Low detection limits are obtained and the linear calibration concentration range of the labels spans 5 or 6 orders of magnitude. The lowest detection limits are obtained with Tb(III) chelates which can be detected down to picomolar levels in aqueous solution using time-resolved measurement techniques. Luminophores, such as, 9-fluorenylmethyl chloroformate, derivatives of fluorescein and its analogues, aromatic lanthanide(III) chelates, various coumarins and porphyrins can be used as labels emitting in different spectral regions. The extraordinary analytical power of the tunnel emission electrodes lies in the possibility of simultaneously exciting several different labels emitting either in the UV, visible or NIR range and luminescence lifetimes varying from the ns to the ms range. Therefore, wavelength or time discrimination or their combination can be exploited in separation of the electrochemiluminescence signals from different labels.
Rhodamine B (RhB) exhibits strong cathodic electrogenerated chemiluminescence (ECL) in aqueous solutions during high-amplitude pulse polarization at thin oxide film-coated aluminum electrodes. This method allows the detection of RhB below nanomolar concentration level and provides linear calibration plots spanning over several orders of magnitude of concentration. In addition, a relatively long ECL lifetime of RhB provides a basis for time-resolved detection. Thus, widely used RhB-based labels can also be suggested to be usable as electrochemiluminescent labels in fully aqueous solutions in bioaffinity assays such as in immunoassays and DNA-probing assays. Support was obtained for the chemiluminescence generation mechanism to be essentially the same as that of radiochemiluminescence in aqueous solution.
A time-resolved electroluminometer, which utilizes electrogenerated luminescence (EL) from disposable oxide-covered aluminium electrodes, is described in detail. The cathodic polarization of this disc electrode in aqueous electrolytes induces a faint background EL with peak emission at 570 nm and with a lifetime of 6 μs. This cell can be utilized to excite dysprosium(III), samarium(III) and terbium(III) by electrochemical means to their lowest excited singlet states and, thus, to produce lanthanide(III)-specific emissions. From the lanthanide(III) El systems tested so far, a cathodically induced EL of 2,6-bis[N,N,bis(carboxymethyl)aminomethyl]-4-benzoylphenol-chelated terbium(III) with a 2.1-ms lifetime is the longest lived and can be efficiently discriminated against the background EL on a time-resolved basis using instrumentally simple time-resolved detection; this time-resolved terbium(III) EL makes possible the determination of terbium(III) even below the picomole level in aqueous solutions.
Hot electrons can be injected from conductor/insulator/electrolyte (C/I/E) junctions into an aqueous electrolyte solution. Injected hot electrons induce electrogenerated chemiluminescence (ECL) of various luminophores in fully aqueous solutions. Such ECL gives the basis of electrochemiluminoimmunoassays and DNA-probe assays where different luminophores can be used as electrochemiluminescent labels. This work shows that SYBR® Green I is suitable as an ECL label in detection methods based on C/I/E tunnel-emission electrodes such as oxide-coated aluminium, magnesium and silicon electrodes.
Cathodic pulse polarisation of thin insulating film-coated electrodes enables the generation of electrochemiluminescence (ECL) by tunnel emission of hot electrons from the Fermi level of the conductor material of the conductor–insulator–aqueous electrolyte solution junction to the solutes at the vicinity of the electrode surface and probably also to the conduction band of water. The latter process can generate hydrated electrons as strongly reducing slightly longer-lived cathodic intermediates, which are known to be able to induce chemiluminescence (CL) of various types of luminophores having very different photophysical and chemical properties. The generation of the above-mentioned cathodic primary species provides good possibilities to use many types of luminophores as label molecules in sensitive immuno and DNA-probing assays. This paper introduces an electrochemiluminoimmunoassay (ECLIA) for human thyroid stimulating hormone (hTSH) at oxide-coated n-silicon electrodes and demonstrates the suitability of silicon electrodes covered with thermally grown silicon dioxide film as disposable working electrodes (WEs) in sensitive time-resolved ECL (tr-ECL) measurements in aqueous solution. The label chelate can be detected almost down to picomolar level and the calibration curve of the chelate covers more than five orders of magnitude of chelate concentration. Also the calibration curve of the immunometric hTSH assay was found to be linear over a wide range of hTSH concentration, the detection limit of the hormone being below 1 mU l−1 (4 pmol l−1).
Chemiluminescence (CL) detection is seldom used in two-dimensional solid support microarray platforms because adequate sensitivity and spatial resolution is difficult to achieve. The three-dimensional ordered microchannels of the Flow-thru Chip increase both the sensitivity and spatial resolution required for quantitative CL measurements on microarrays. Enzyme-catalyzed CL reactions for the detection of hybridizations on microchannel glass were imaged using a CCD camera. Signal uniformity, sensitivity, and dynamic range of the detection method were determined. The relative standard deviation of signal intensities across an array of 64 spots was 8.1%. A detection limit of 250 amol of target with a linear dynamic range of 3 orders of magnitude was obtained for a 3-h assay. Similar to two-color fluorescence measurements, multiple enzyme labels were employed to demonstrate two-channel chemiluminescence. A unique method for measuring the relaxation time of a chemiluminescent species is also described.
The rate of hybridization of oligonucleotide target sequences to chemically immobilized oligonucleotide probes has been studied both with and without an electrical field. The probe size was 20-24 nucleotides (nt) while the target size ranged from 157 to 864 nt. In agreement with previous studies, complete hybridization under normal conditions required 10-30 hours, depending on target size. The kinetics were characterized by a characteristic lag time followed by an asymptotic rise to the final value. In contrast, with an applied electrical field, all but the largest target hybridized in about 10 min while the longest hybridized within 1 h. Deleterious electrode reactions were avoided by close spacing of the anode and cathode and application of very small voltages. Our results suggest that probes and targets orient flat on the surface. A model is suggested to explain the kinetics observed that involves a series of surface states between initial target arrival and final hybridized state. Our results show that the electric field accelerated hybrid capture of solution-phase targets by surface-bound probes. This approach may have implications for enhancing array-based hybrid capture for mutation detection, copy number determination and/or gene expression profiling.
We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR.
Anodic electrogenerated chemiluminescence (ECL) with tri-n-propylamine (TPrA) as a coreactant was used to determine DNA and C-reactive protein (CRP) by immobilizations on Au(111) electrodes using tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) labels. A 23-mer synthetic single-stranded (ss) DNA derived from the Bacillus anthracis with an amino-modified group at the 5' end position was covalently attached to the Au(111) substrate precoated with a self-assembled thiol monolayer of 3-mercaptopropanoic acid (3-MPA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and then hybridized with a target ssDNA tagged with Ru(bpy)(3)(2+) ECL labels. Similarly, biotinylated anti-CRP species were immobilized effectively onto the Au(111) substrate precovered with a layer of avidin linked covalently via the reaction between avidin and a mixed thiol monolayer of 3-MPA and 16-mercaptohexadecanoic acid on Au(111) in the presence of EDAC and N-hydroxysuccinimide. CRP and anti-CRP tagged with Ru(bpy)(3)(2+) labels were then conjugated to the surface layer. ECL responses were generated from the modified electrodes described above by immersing them in a TPrA-containing electrolyte solution. A series of electrode treatments, including blocking free -COOH groups with ethanol amine, pinhole blocking with bovine serum albumin, washing with EDTA/NaCl/Tris buffer, and spraying with inert gases, were used to reduce the nonspecific adsorption of the labeled species. The ECL peak intensity was linearly proportional to the analyte CRP concentration over the range 1-24 microg/mL. CRP concentrations of two unknown human plasma/serum specimens were measured by the standard addition method based on this technique.
Many genomic assays rely on a distance-dependent interaction between luminescent labels, such as luminescence quenching or resonance energy transfer. We studied the interaction between electrochemically excited Ru(bpy)(3) (2+) and Cy5 in a hybridization assay on a chip. The 3' end of an oligonucleotide was labelled with Ru(bpy)(3) (2+) and the 5' end of a complementary strand with Cy5. Upon the hybridization, the electrochemiluminescence (ECL) of Ru(bpy)(3) (2+) was efficiently quenched by Cy5 with a sensitivity down to 30 nmol/L of the Cy5-labelled complementary strand. The quenching efficiency is calculated to be 78%. A similar phenomenon was observed in a comparative study using laser-excitation of Ru(bpy)(3) (2+). The hybridization with the non-labelled complementary or labelled non-complementary strand did not change the intensity of the ECL signal. Resonance energy transfer, electron transfer and static quenching mechanisms are discussed. Our results suggest that static quenching and/or electron transfer are the most likely quenching mechanisms.
Electrochemical DNA detection systems are an attractive approach to the development of multiplexed, high-throughput DNA analysis systems for clinical and research applications. We have engineered a new class of nanoelectrode ensembles (NEEs) that constitute a useful platform for biomolecular electrochemical sensing. High-sensitivity DNA detection was achieved at oligonucleotide-functionalized NEEs using a label-free electrocatalytic assay. Attomole levels of DNA were detected using the NEEs, validating the promise of nanoarchitectures for ultrasensitive biosensing.
A versatile method for direct, covalent attachment of DNA microarrays at silicon nitride layers, previously deposited by chemical vapor deposition at silicon wafer substrates, is reported. Each microarray fabrication process step, from silicon nitride substrate deposition, surface cleaning, amino-silanation, and attachment of a homobifunctional cross-linking molecule to covalent immobilization of probe oligonucleotides, is defined, characterized, and optimized to yield consistent probe microarray quality, homogeneity, and probe-target hybridization performance. The developed microarray fabrication methodology provides excellent (high signal-to-background ratio) and reproducible responsivity to target oligonucleotide hybridization with a rugged chemical stability that permits exposure of arrays to stringent pre- and posthybridization wash conditions through many sustained cycles of reuse. Overall, the achieved performance features compare very favorably with those of more mature glass based microarrays. It is proposed that this DNA microarray fabrication strategy has the potential to provide a viable route toward the successful realization of future integrated DNA biochips.
The assembly of two aminosilanes on silicon dioxide surfaces is investigated in this work. It is found that for 3-aminopropyltrimethoxysilane (APS), a smaller concentration of the silane and trace amounts of water in the deposition medium, an optimum time, and a postdeposition thermal curing are necessary to obtain a high primary-amine content. By optimization of deposition conditions, uniform APS films with a primary-amine content of 88.6% were obtained. The dependence of the primary-amine content on the experimental parameters is related to the extent to which amines are lost to hydrogen bonding with each other or with the substrate surface. Whenp-aminophenyltrimethoxysilane (APhS) was used, the primary-amine content in the film reached 100% and the surface morphology was more uniform than that of APS films under the same conditions. This is attributed to the rigid phenyl component in APhS that reduces opportunities for hydrogen bonding. In a comparison of the immobilization capacities of the different aminosilane substrates for pyromellitic dianhydride (PMDA), it is observed that higher primary-amine content favors higher uptake, and the APhS film yields 100% PMDA coverage. We infer that primary-amine content could be a measure of the film morphology and accessibility of the substrate amine groups.
The aim of this work was to develop an integrated solution to DNA hybridisation monitoring for diagnostics on a monolithic silicon platform. A fabrication process was developed incorporating a gold initiation electrode patterned directly onto a PIN photodiode detector. Patterned interdigitated type electrodes exhibited the smallest reduction in photodiode sensitivity, therefore these were chosen as the ECL initiator design. A novel DNA hybridisation assay was developed based on the displacement of a partially mismatched complementary strand by a perfectly matched labelled complementary strand. Pre-hybridised thiolated oligonucleotide and unlabelled 25% mismatched oligonucleotide were assembled on the gold initiation electrode. On addition of the labelled perfectly complementary oligonucleotide, the mismatched strands were displaced and a signal was generated. The sensitivity of the photodiode to light emitted at 620 nm, the ruthenium emission wavelength, was determined and subsequently, the diode current response to light generated by flow addition of ruthenium solution was found to be measurable to a concentration of 10 fM. Pre-hybridised duplex DNA, consisting of thiolated oligonucleotide and ruthenium labelled complementary oligonucleotide, was assembled on the gold initiation electrode. The difference between the current measured during flow of buffer and the ECL co-reactant TPA was three orders of magnitude, indicating that DNA assembled on the surface comprised sufficient ruthenium to generate a measurable signal. Finally, the displacement of unlabelled partial mismatch oligonucleotide from the sensor surface was monitored on addition of the ruthenium labelled perfectly complementary oligonucleotide in TPA flow and the measured photodiode current response was up to 50 times greater.
Describes the detection and quantification of DNA strands using an electrochemiluminescence (ECL) instrument with microfabricated excitation/detection cell. The microcell contains thin film Pt electrodes, fluid microchannels and a silicon PIN diode photodetector. The DNA strands are labeled with an electrochemically exited, luminescing molecule: tris( 2, 2' bipyridyl) ruthenium (TBR). In order to separate the free label from labeled DNA, the DNA strands are attached to paramagnetic beads with a biotin-streptavidin linkage. With a 1 cm2 Pt anode, a detection limit of 40 femtomoles of DNA is obtained with a silicon PIN diode photodetector operating at room temperature. The overall size of the microcell is only 1.5×2×0.3 cm with a sample volume of 150 μL
- Y Okahata
- Y Matsunobu
- K Ijiro
- M Mukae
- A Murakami
- K Makino
Y. Okahata, Y. Matsunobu, K. Ijiro, M. Mukae, A. Murakami, K.
Makino, J. Am. Chem. Soc. 114 (1992) 8299.
- G F Blackburn
- H P Shah
- J H Kenten
- J Leland
- R A Kamin
- J Link
- J Peterman
- M J Powell
- A Shah
- D B Talley
- S K Tyagi
- E Wilkins
- T G Wu
- R J Massey
G.F. Blackburn, H.P. Shah, J.H. Kenten, J. Leland, R.A. Kamin, J. Link,
J. Peterman, M.J. Powell, A. Shah, D.B. Talley, S.K. Tyagi, E. Wilkins,
T.G. Wu, R.J. Massey, Clin. Chem. 37 (1991) 1534.
- J Suomi
- M Hakansson
- Q Jiang
- M Kotiranta
- M Helin
- A J Niskanen
- S Kulmala
J. Suomi, M. Hakansson, Q. Jiang, M. Kotiranta, M. Helin, A.J. Niskanen, S. Kulmala, Electrochim. Acta 541 (2005) 165.
- A.-M Spehar
- S Koster
- S Kulmala
- E Verpoorte
- N De Rooij
- M Koudelka-Hep
A.-M. Spehar, S. Koster, S. Kulmala, E. Verpoorte, N. de Rooij, M.
Koudelka-Hep, Luminescence 19 (2004) 287.
- E Souteyrand
- J P Cloarec
- J R Martin
- C Wilson
- I Lawrence
- S Mikkelsen
- M F F Lawrence
- M P Zhang
- M Srinivasan
- G Manning
- Redmond
E. Souteyrand, J.P. Cloarec, J.R. Martin, C. Wilson, I. Lawrence, S.
Mikkelsen, M.F. Lawrence, J. Phys. Chem. B 101 (1997) 2980.
[29] F. Zhang, M.P. Srinivasan, Langmuir 20 (2004) 2309.
[30] M. Manning, G. Redmond, Langmuir 21 (2005) 395.
[31] H.-J. Su, S. Surrey, S.E. McKenzie, P. Fortina, D.J. Graves, Electrophoresis 23 (2002) 1551.
- J Kankare
- K Haapakka
- S Kulmala
- V Nanto
- J Eskola
- H Takalo
J. Kankare, K. Haapakka, S. Kulmala, V. Nanto, J. Eskola, H. Takalo,
Anal. Chim. Acta 266 (1992) 205.
- Y.-T Hsueh
- S Collins
- R L Smith
Y.-T. Hsueh, S. Collins, R.L. Smith, Sens. Actuators B 49 (1998) 1.
- L A Chrisey
- G U Lee
L.A. Chrisey, G.U. Lee, C.E. O'Ferrall, Nucleic Acids Res. 24 (1996)
3031.
- R Gasparac
- B J Taft
- M.-A Lapierre-Delvin
- A D Lazareck
- J M Xu
- S O Kelley
R. Gasparac, B.J. Taft, M.-A. Lapierre-Delvin, A.D. Lazareck, J.M. Xu,
S.O. Kelley, J. Am. Chem. Soc. 126 (2004) 12270.
- W Miao
- A J Bard
W. Miao, A.J. Bard, Anal. Chem. 75 (2003) 5825.
- A V Saprigin
- C W Thomas
- C S Dulcey
- C H Patterson Jr
- M S Spector
A.V. Saprigin, C.W. Thomas, C.S. Dulcey, C.H. Patterson Jr., M.S. Spector, Surf. Interface Anal. 36 (2004) 24.
- D G Kurth
- T Bein
D.G. Kurth, T. Bein, Langmuir 11 (1995) 3061.
- S J Oh
- S J Cho
- C O Kim
- J W Park
S.J. Oh, S.J. Cho, C.O. Kim, J.W. Park, Langmuir 18 (2002) 1764.
- P T Charles
- G J Vora
- J D Andreadis
- A J Fortney
- C E Meador
- C S Dulcey
- D A Stenger
P.T. Charles, G.J. Vora, J.D. Andreadis, A.J. Fortney, C.E. Meador, C.S.
Dulcey, D.A. Stenger, Langmuir 19 (2003) 1586.
- M Helin
- Q Jiang
- H Ketamo
- M Hakansson
- A.-M Spehar
- S Kulmala
- T Ala-Kleme
M. Helin, Q. Jiang, H. Ketamo, M. Hakansson, A.-M. Spehar, S. Kulmala, T. Ala-Kleme, Electrochim. Acta 51 (2005) 725.