Determination of Dansyl Amino Acids and Oxalate by HPLC with Electrogenerated Chemiluminescence Detection Using Tris(2,2'-bipyridyl)ruthenium(II) in the Mobile Phase

A new electrogenerated chemiluminescence detection method is investigated for use in detection in reversed-phase and reversed-phase ion-pair HPLC with Ru(bpy)(3)(2+) in the mobile phase. In this method, different concentrations of Ru(bpy)(3)(2+) are dissolved in the mobile phase and the HPLC column flushed with the mobile phase for 1 h until the column is saturated with Ru(bpy)(3)(2+). The separated analytes along with Ru(bpy)(3)(2+) pass through an optical-electrochemical flow cell which has a dual platinum electrode held at a potential of 1250 mV vs a Ag/AgCl reference electrode. On the surface of the electrode, Ru(bpy)(3)(2+) is oxidized to Ru(bpy)(3)(3+) which reacts with the analytes to emit light. The retention times, retention orders, detection limits, and linearity in working curves are compared to those obtained with the conventional postcolumn Ru(bpy)(3)(2+) addition method. The retention times for dansyl amino acids with Ru(bpy)(3)(2+) in the mobile phase are longer than those obtained with the postcolumn addition approach. This may be caused by π-to-π interactions between the aromatic groups of the dansyl derivatives and the bipyridyl groups of Ru(bpy)(3)(2+) in the Ru(bpy)(3)(2+)-saturated reversed-phase column. Similarly, oxalate is separated from urine and blood plasma samples by reversed-phase ion-pair HPLC. Plasma samples are obtained using ultrafiltration to remove proteins from whole blood. Retention times for oxalate with the two detection techniques are identical, and detection limits for these techniques are compared.     

A single calibration graph for the direct determination of ascorbic and dehydroascorbic acids by electrogenerated luminescence based on Ru(bpy)(3)2+ in aqueous solution

Ascorbic (H2A) and dehydroascorbic (DA) acids were for the first time directly determined in a single chromatographic run by means of the tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)2+) based electrogenerated chemiluminescence (ECL) detection. For the first time, it was demonstrated that DA, a nonelectroactive compound, is ECL active and is responsible for the ECL behavior of H2A. This fact, together with the lack of a DA standard, suggested the use of a calibration graph obtained for H2A, for determining both analytes. The proven ECL activity of DA, together with literature data relative to the standard redox potentials of the different species coming from H2A, led to a reconsideration of the proposed ECL reaction mechanism for H2A. The role of the OH- ion in the reaction mechanism of the two analytes appeared to be crucial. H2A and DA could be separated by a suitable C18-reversed-phase HPLC column using an aqueous 30 mM H3PO4 solution as the mobile phase. The optimal ECL response was achieved by polarizing the working electrode at 1.150 Vvs SCE (standard calomel electrode) (oxidation diffusion limiting potential for both H2A and Ru(bpy)(3)2+). The Ru(bpy)(3)2+ solution, at pH 10 for carbonate buffer, was mixed to the eluent solution in a postcolumn system, obtaining, still at pH 10, the final 0.25 mM Ru(bpy)(3)2+ concentration. The detection limit found for the two analytes was 1 x 10(-7) M. The method was successfully applied to the determination of the analytes in a commercially available orange fruit juice.     

Method for effective immobilization of Ru(bpy)(3)2+ on an electrode surface for solid-state electrochemiluminescene detection

A novel method for effective immobilization of Ru(bpy)3(2+) on an electrode surface is developed. The whole process involves two steps: the electrostatic interactions between citrate-capped gold nanoparticles (AuNPs) and Ru(bpy)3Cl2 in aqueous medium were used to fabricate Ru(bpy)(3)2+-AuNP aggregates (Ru-AuNPs) first, and then the Au-S interactions between as-formed Ru-AuNPs and sulfhydryl groups were used to effectively immobilize the Ru-AuNPs on a sulfhydryl-derivated indium tin oxide (ITO) electrode surface. As-prepared ITO electrode shows excellent stability, and the ECL active species Ru(bpy)3(2+) contained therein exhibit excellent ECL behaviors.     

Multilabeling biomolecules at a single site. 1. Synthesis and characterization of a dendritic label for electrochemiluminescence assays

Ultrasensitive bioanalytical assays are of great value for early detection of human diseases and pathogens. The sensitivities of immunoassays and DNA probing can be enhanced by multilabeling the biorecognition partner used for affinity-based assays. However, the bioreactivity of biomolecules is affected by a high degree of multilabeling at multiple functional sites. It is proposed that dendritic scaffoldings be used to link multiple signal-generating units to a single site with potentially minimum impact on the bioaffinity. A prototype label, a zeroth-generation dendron, bearing three [Ru(bpy)(3)](2+) units for electrochemiluminescence (ECL) assays was synthesized and characterized preliminarily by spectroscopic, electrochemical, and ECL methods. No evidence of interaction between the neighboring [Ru(bpy)(3)](2+) units in the label molecule was found from these characterizations. Both the photoluminescence and ECL of the prototype label have features very similar to those of mononuclear [Ru(bpy)(3)](2+) compounds. Labeling a model protein with a triad of [Ru(bpy)(3)](2+) at one NH(2) position was demonstrated. The results reported here provide support to applying the proposed multilabeling strategy to affinity-based bioanalytical assays.     

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 2. Demonstration of selectivity in the presence of direct interferences

Three modes of selectivity based on charge-selective partitioning, electrolysis potential, and spectral absorption wavelength were demonstrated simultaneously in a new type of spectroelectrochemical sensor. Operation and performance of the three modes of selectivity for detection of analytes in the presence of direct interferences were investigated using binary mixture systems. These binary mixtures consisted of Fe(CN)(6)(3-) and Ru(bpy)(3)(2+) and of Fe(CN)(6)(4-) and Ru(CN)(6)(4)(-) in aqueous solutions. Results on the Fe(CN)(6)(3-)/Ru(bpy)(3)(2+) binary mixture showed that an anion-exchange coating consisting of PDMDAAC-SiO(2) [where PDMDAAC is poly(dimethyldiallylammonium chloride)] and a cation-exchange coating consisting of Nafion-SiO(2) can trap and preconcentrate analytes with charge selection. At the same time, such coatings exclude interferences carrying the same type of charge as that of the exchange sites in the sensor coating. Using the Fe(CN)(6)(4-)/Ru(CN)(6)(4-) binary mixture, the Fe(CN)(6)(4-) component can be selectively detected by restricting the modulation potential cycled to a range specific to the redox-active Fe(CN)(6)(4-) component and simultaneously monitoring the optical response at the overlapping wavelength of 420 nm. It was also shown that, when the wavelength for optical monitoring was chosen as 500 nm, which is specific to the Ru(CN)(6)(4-) component, interference from the Fe(CN)(6)(4-) component for spectroelectrochemical detection of Ru(CN)(6)(4-) was significantly suppressed, even though the cyclic modulation potential encompassed the redox range for the Fe(CN)(6)(4-) component.     

A two-channel microfluidic sensor that uses anodic electrogenerated chemiluminescence as a photonic reporter of cathodic redox reactions

This paper describes a new approach for sensing electrochemically active substrates in microfluidic systems. This two-electrode sensor relies on electrochemical detection at one electrode and electrogenerated chemiluminescent (ECL) reporting at the other. Each microfabricated indium tin oxide electrode is located in a separate microfluidic channel, but the channels are connected downstream of the electrodes to maintain a complete electrical circuit. Because of laminar flow, there is no bulk mixing of the fluids in the detecting and reporting channels. This approach allows the ECL reaction to be physically and chemically decoupled from the sensing channel of the device, which greatly expands the number of analytes that can be detected. However, because the cathode and anode are connected, electron-transfer processes occurring at the sensing electrode are electrically coupled to the ECL reaction. Charge balance permits the ECL light output to be quantitatively correlated to electrochemical reductions at the cathode. The system is used to detect Fe(CN)6(3-), Ru(NH3)6(3+), and benzyl viologen and report their presence via Ru(bpy)3(2+) (bpy = bipyridine) luminescence. Each different redox target initiates ECL at a unique potential bias related to its standard redox potential. The influence of the concentrations of Ru(bpy)3(2+) and the target analytes is discussed.     

Inhibited Ru(bpy)3 2+ electrochemiluminescence related to electrochemical oxidation activity of inhibitors

Electrochemiluminescence (ECL) has been accepted by the analytical chemist as a powerful tool for detection of many inorganic and organic compounds. Ru(bpy)3 2+ has been the most popular ECL system, and many investigations have been focused on the application based on the enhancement or inhibition of Ru(bpy)3 2+ ECL system. However, not much attention has been paid to the theoretical investigation of this ECL system, especially to the inhibiting mechanism for the Ru(bpy)3 2+ ECL system. In the present study, many of the inorganic and organic compounds with electrochemical oxidation activity were found to strongly inhibit Ru(bpy)3 2+ ECL. To explain these inhibited ECL phenomena, a new "electrochemical oxidation inhibiting" mechanism has been proposed via the establishment of a corresponding model. The effects of applied potential, uncompensated resistance, and concentration of inhibitor on the inhibited ECL derived from the model have been verified by experiments. The new ECL inhibition mechanism can be commonly used to explain many kinds of inhibited ECL presently observed, and it is envisioned to result in finding of more inhibitors of this type and establishment of new sensitive ECL detection methods for them.     

Electrochemiluminescence of Tris(2,2'-bipyridine)ruthenium in Water at Carbon Microelectrodes

Electrochemiluminescence (ECL) of Ru(bpy)(3)(2+) in water only, without any added electrolyte or reducing agents, has been obtained at carbon interdigitated microelectrode arrays (C-IDAs) of 2 μm width and spacing. In a generation/collection biasing mode, ECL can be clearly seen with the naked eye in normal room lighting at concentrations greater than 1 mM. Using a conventional photomultiplier tube (PMT), a detection limit of 10(-)(7) M Ru(bpy)(3)(2+) has been achieved for an electrode area of 0.25 mm(2). In comparison, the ECL intensity produced at Pt-IDA of the same geometry, under identical experimental conditions, was more than 300 times less. The ECL obtained at C-IDAs is attributed to the annihilation reaction of the reduced and oxidized forms of the Ru(bpy)(3)(2+) made possible due to the small electrode spacing.     

4-(Dimethylamino)butyric acid labeling for electrochemiluminescence detection of biological substances by increasing sensitivity with gold nanoparticle amplification

4-(Dimethylamino)butyric acid (DMBA) labeling combined with gold nanoparticle amplification for electrochemiluminescence (ECL) determination of a biological substance (bovine serum albumin (BSA) and immunoglobulin G (IgG) as models) was presented. After DMBA, an analogue of tripropylamine, was tagged on the (anti)analytes, an ECL signal related to the content of the analytes was generated when the analyte tagged with DMBA was in contact with tris(2,2'-bipyridine)ruthenium (Ru(bpy)(3)2+) solution and a potential was applied. To improve the adsorption capacity, a gold nanoparticle layer was first combined into the surface of the 2-mm-diameter gold electrode. For the determination of BSA, avidin was covalently conjugated to a self-assembled monolayer of 3-mercaptopropanoic acid on the gold nanoparticle layer. Biotinylated BSA-DMBA was then immobilized on the gold nanoparticle layer of the gold electrode via the avidin-biotin reaction. IgG was tested via a typical sandwich-type immobilization method. ECL signals were generated from the electrodes immobilized with BSA or IgG by immersing them in a 1 mmol L-1 Ru(bpy)(3)2+ solution and scanning from 0.5 to 1.3 V versus Ag/AgCl. With gold nanoparticle amplification, the ECL peak intensity was proportional to the concentration over the range 1-80 and 5-100 microg/mL for BSA and IgG consuming 50 microL of sample, respectively. A 10- and 6-fold sensitivity enhancement was obtained for BSA and IgG over their direct immobilization on an electrode using DMBA labeling. The relative standard deviations of five replicate determinations of 10 microg/mL BSA and 20 microg/mL IgG were 8.4 and 10.2%, respectively. High biocompatibility and low cost were the main advantages of the present DMBA labeling technique over the traditional Ru(bpy)(3)2+ labeling.     

Selective photoelectrochemical detection of DNA with high-affinity metallointercalator and tin oxide nanoparticle electrode

Selective detection of double-stranded DNA (ds-DNA) in solution was achieved by photoelectrochemistry using a high-affinity DNA intercalator, Ru(bpy)2dppz (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine) as the signal indicator and tin oxide nanoparticle as electrode material. When Ru(bpy)2dppz alone was irradiated with 470-nm light, anodic photocurrent was detected on the semiconductor electrode due to electron injection from its excited state into the conduction band of the electrode. The current was sustained in the presence of oxalate in solution, which acted as a sacrificial electron donor to regenerate the ground-state metal complex. After addition of double-stranded calf thymus DNA into the solution, photocurrent dropped substantially. The drop was attributed to the intercalation of Ru(bpy)2dppz into DNA and, consequently, the reduced mass diffusion of the indicator to the electrode, as well as electrostatic repulsion between oxalate anion and negative charges on DNA. The degree of signal reduction was a function of the DNA concentration, thus forming the basis for real-time DNA detection. The signal reduction was selective for ds-DNA, as no such effect was observed for single-stranded polynucleotides such as poly-G, poly-C, poly-A, and poly-U. The detection limit of calf thymus ds-DNA reached 1.8 x 10(-10) M in solution.     

Spectroelectrochemical sensing based on multimode selectivity simultaneously achievable in a single device. 16. Sensing by fluorescence

A fluorescence spectroelectrochemical sensor capable of detecting very low concentrations of metal complexes is described. The sensor is based on a novel spectroelectrochemical sensor that incorporates multiple internal reflection spectroscopy at an optically transparent electrode (OTE) coated with a selective film to enhance detection limits by preconcentrating the analyte at the OTE surface. Nafion was used as the selective cation exchange film for detecting Ru(bpy)(3)(2+), the model analyte, which fluoresces at 605 nm when excited with a 441.6-nm HeCd laser. The unoptimized linear dynamic range of the sensor for Ru(bpy)(3)(2+) is between 1 x 10(-)(11) and 1 x 10(-)(7) M with a calculated 2 x 10(-)(13) M detection limit. The sensor employs extremely thin films ( approximately 12 nm) without significantly sacrificing its sensitivity. The sensor response is demonstrated with varying film thicknesses. A state-of-the-art flow cell design allows variable cell volumes as low as approximately 4 microL. Fluorescence of the sample can be controlled by electromodulation between 0.7 and 1.3 V. Sensor operation is not reversible for the chosen model film (Nafion) and sample (Ru(bpy)(3)(2+)) but it can be regenerated with ethanol for multiple uses.     

Stable tris(2,2'-bipyridine)ruthenium(III) for chemiluminescence detection

Two exceedingly stable [Ru(bipy)(3)](3+) reagents were prepared by dissolving either [Ru(bipy)(3)](ClO(4))(2) in acetonitrile (containing 0.05 M HClO(4)) or [Ru(bipy)(3)]Cl(2)·6H(2)O in 95:5 glacial acetic acid-acetic anhydride (containing 0.05 M H(2)SO(4)) followed by oxidation with PbO(2). These conveniently prepared solutions provide highly reproducible chemiluminescence detection over long periods of analysis, avoiding the need for recalibration or preparation of fresh reagent solutions and without the complications associated with online chemical or electrochemical oxidations. The reagent prepared in acetonitrile produced much greater signal intensities with a range of analytes and was deemed most suitable for high-performance liquid chromatography (HPLC) with postcolumn chemiluminescence detection.     

Quenching of electrogenerated chemiluminescence by phenols, hydroquinones, catechols, and benzoquinones

Efficient quenching of Ru(bpy)(3)(2+) (bpy = 2,2'-bipyridine) electrogenerated chemiluminescence has been observed in the presence of phenols, catechols, hydroquinones, and benzoquinones. In most instances, quenching is observed with 100-fold excess of quencher over Ru(bpy)(3)(2+), with complete quenching observed between 1000- and 2000-fold excess. The mechanism of quenching is believed to involve energy transfer from the excited-state luminophore to benzoquinone. In the case of phenols, catechols, and hydroquinones, quenching is believed to occur via a benzoquinone derivative formed at the electrode surface. Photoluminescence and UV-visible experiments coupled with bulk electrolysis support the formation of benzoquinone products upon electrochemical oxidation.     

Chemiluminescence detection using regenerable tris(2,2'-bipyridyl)ruthenium(II) immobilized in Nafion.

The development of a detection method based on the electrogenerated chemiluminescence of tris(2,2'-bipyridine)ruthenium(II), (Ru(bpy)3(2+], immobilized in a Nafion film coated on an electrode is discussed. Control of the electrode potential controls creation of the reactive reagent Ru(bpy)3(3+) which reacts with certain analytes to yield chemiluminescence emission of intensity proportional to the analyte concentration. The reaction results in Ru(bpy)3(3+) being converted to Ru(bpy)3(2+), which then is recycled to Ru(bpy)3(3+) again at the electrode. This sensor has been used in flow injection to determine oxalate, alkylamines, and NADH. Detection limits are 1 microM, 10 nM, and 1 microM, respectively, with working ranges extending over 4 decades in concentration. Sensitivity is constant over the wide pH range from 3 to 10. With oxalate, and to a small extent with amines, emission intensities increase with increasing ionic strength; this was shown to be a phenomenon related to the Nafion film and not to the chemiluminescence reaction. Emission intensities increase with temperature. The sensor remains stable for several days with suitable storage conditions. Significant amounts of Ru(bpy)3(3+) are shown to be capable of storage within the film.     

Electrogenerated chemiluminescence from tris(2,2'-bipyridyl)ruthenium(II) immobilized in titania-perfluorosulfonated ionomer composite films

Electrochemical behavior and electrogenerated chemiluminescence (ECL) of tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)3(2+)) immobilized in sol-gel-derived titania TiO2)-Nafion composite films coated on a glassy carbon electrode have been investigated. The electroactivity of Ru(bpy)3(2+) ion exchanged into the composite films and its ECL behavior were strongly dependent upon the amount of Nafion incorporated into the TiO2-Nafion composite films. The ECL sensor of Ru(bpy)32+ immobilized in a TiO2-Nafion composite with 50% Nafion content showed the maximum ECL intensities for both tripropylamine (TPA) and sodium oxalate in 0.05 M phosphate buffer solution at pH 7. Detection limits were 0.1 microM for TPA and 1.0 microM for oxalate (S/N = 3) with a linear range of 3 orders of magnitude in concentration. The present ECL sensor showed improved ECL sensitivity and long-term stability, as compared to the ECL sensors based on pure Nafion films. The present Ru(bpy)3(2+) ECL sensor based on TiO2-Nafion (50%) composite films was applied as an HPLC detector for the determination of erythromycin in human urine samples. The present Ru(bpy)3(2+) ECL sensor was stable in the mobile phase containing a high content of organic solvent (30%, v/v), in contrast to a pure Nafion-based Ru(bpy)3(2+) ECL sensor. The detection limit for erythromycin was 1.0 microM, with a linear range of 3 orders of magnitude in concentration.     

Electrogenerated chemiluminescence from R(bpy)3(2+) ion-exchanged in carbon nanotube/perfluorosulfonated ionomer composite films

The electrochemistry and electrogenerated chemiluminescence (ECL) of ruthenium(II) tris(bipyridine) (Ru(bpy)(3)(2+)) ion-exchanged in carbon nanotube (CNT)/Nafion composite films were investigated with tripropylamine (TPA) as a coreactant at a glassy carbon (GC) electrode. The major goal of this work was to investigate and develop new materials and immobilization approaches for the fabrication of ECL-based sensors with improved sensitivity, reactivity, and long-term stability. Ru(bpy)(3)(2+) could be strongly incorporated into Nafion film, but the rate of charge transfer was relative slow and its stability was also problematic. The interfusion of CNT in Nafion resulted in a high peak current of Ru(bpy)(3)(2+) and high ECL intensity. The results indicated that the composite film had more open structures and a larger surface area allowing faster diffusion of Ru(bpy)(3)(2+) and that the CNT could adsorb Ru(bpy)(3)(2+) and also acted as conducting pathways to connect Ru(bpy)(3)(2+) sites to the electrode. In the present work, the sensitivity of the ECL system at the CNT/Nafion film-modified electrodes was more than 2 orders of magnitude higher than that observed at a silica/Nafion composite film-modified electrode and 3 orders of magnitude higher than that at pure Nafion films. The CNT/Nafion composite film-modified GC electrodes also exhibited long-term stability.     

Electrogenerated chemiluminescence. 72. Determination of immobilized DNA and C-reactive protein on Au(111) electrodes using tris(2,2'-bipyridyl)ruthenium(II) labels

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.     

Pt nanoparticles: Heat treatment-based preparation and Ru(bpy)(3)2+-mediated formation of aggregates that can form stable films on bare solid electrode surfaces for solid-state electrochemiluminescence detection

A simple thermal process for the preparation of small Pt nanoparticles is presented, carried out by heating a H2PtCl6/3-thiophenemalonic acid aqueous solution. The following treatment of such colloidal Pt solution with Ru(bpy)(3)2+ causes the assembly of Pt nanoparticles into aggregates. Most importantly, directly placing such aggregates on bare solid electrode surfaces can produce very stable films exhibiting excellent electrochemiluminescence behaviors.     

Sensitive electrochemiluminescence detection of c-Myc mRNA in breast cancer cells on a wireless bipolar electrode

We report an ultrasensitive wireless electrochemiluminescence (ECL) protocol for the detection of a nucleic acid target in tumor cells on an indium tin oxide bipolar electrode (BPE) in a poly(dimethylsiloxane) microchannel. The approach is based on the modification of the anodic pole of the BPE with antisense DNA as the recognition element, Ru(bpy)(3)(2+)-conjugated silica nanoparticles (RuSi@Ru(bpy)(3)(2+)) as the signal amplification tag, and reporter DNA as a reference standard. It employs the hybridization-induced changes of RuSi@Ru(bpy)(3)(2+) ECL efficiency for the specific detection of reporter DNA released from tumor cells. Prior to ECL detection, tumor cells are transfected with CdSe@ZnS quantum dot (QD)-antisense DNA/reporter DNA conjugates. Upon the selective binding of antisense DNA probes to intracellular target mRNA, reporter DNA will be released from the QDs, which indicates the amount of the target mRNA. The proof of concept is demonstrated using a proto-oncogene c-Myc mRNA in MCF-7 cells (breast cancer cell line) as a model target. The wireless ECL biosensor exhibited excellent ECL signals which showed a good linear range over 2 × 10(-16) to 1 × 10(-11) M toward the reporter DNA detection and could accurately quantify c-Myc mRNA copy numbers in living cells. C-Myc mRNA in each MCF-7 cell and LO2 cell was estimated to be 2203 and 13 copies, respectively. This wireless ECL strategy provides great promise in a miniaturized device and may facilitate the achievement of point of care testing.     

Electrogenerated chemiluminescence detection in paper-based microfluidic sensors

This paper describes the first approach at combining paper microfluidics with electrochemiluminescent (ECL) detection. Inkjet printing is used to produce paper microfluidic substrates which are combined with screen-printed electrodes (SPEs) to create simple, cheap, disposable sensors which can be read without a traditional photodetector. The sensing mechanism is based on the orange luminescence due to the ECL reaction of tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3)(2+)) with certain analytes. Using a conventional photodetector, 2-(dibutylamino)ethanol (DBAE) and nicotinamide adenine dinucleotide (NADH) could be detected to levels of 0.9 μM and 72 μM, respectively. Significantly, a mobile camera phone can also be used to detect the luminescence from the sensors. By analyzing the red pixel intensity in digital images of the ECL emission, a calibration curve was constructed demonstrating that DBAE could be detected to levels of 250 μM using the phone.