Electrochemiluminescent disposable cholesterol biosensor based on avidin-biotin assembling with the electroformed luminescent conducting polymer poly(luminol-biotinylated pyrrole).

An electrochemiluminescent cholesterol disposable biosensor has been prepared by the formation of assembled layers on gold screen-printed cells. The detection layer is based on the electro-formation of new luminol copolymers with different synthesized biotinylated pyrroles prepared by click-chemistry, offering a new transduction layer with new electroluminescent properties on biosensors. The electrochemiluminescence (ECL) luminol copolymers are electroformed by cyclic voltammetry (five cycles) at pH 7.0 uses a10(-3)M biotinylated pyrrole-luminol ratio of 1:10 in PBS buffer. With respect to the recognition layer, cholesterol oxidase was biotinylated by incubation with biotin vinyl sulfone, and immobilized on the copolymer by avidin-biotin interaction. The analytical signal of the biosensor is the ECL enzymatic initial rate working in chronoamperometric mode at 0.5V excitation potential with 10s between pulses at pH 9.5. The disposable device offers a cholesterol linear range from 1.5×10(-5)M to 8.0×10(-4)M with a limit of detection of 1.47×10(-5)M and accuracy of 7.9% for 9.0×10(-5)M and 14.1% for 2.0×10(-4)M, (n=5). Satisfactory results were obtained for cholesterol determination in serum samples compared to a reference procedure.

SPE biosensor for cholesterol in serum samples based on electrochemiluminescent luminol copolymer.

A poly(luminol-3,3',5,5'-tetramethylbenzidine) copolymer manufactured by electropolymerization on screen-printed gold electrodes greatly improves the electrochemiluminescence of hydrogen peroxide. Cholesterol oxidase was immobilized on the surface of a poly(luminol-3,3',5,5'-tetramethylbenzidine) screen-printed cell modified with chitosan to prepare an ECL biosensor for cholesterol. Working under the optimized conditions, the linear dynamic range of cholesterol was 2.4 × 10(-5)-1.0 × 10(-3)M with a limit of detection of 7.3 × 10(-6)M and a precision of 10.3% (5.0 × 10(-4)M, n=5) expressed as relative standard deviation. This biosensor was applied to the determination of total cholesterol in serum samples obtaining satisfactory results with respect to the reference procedure. This cholesterol biosensor offers an alternative analytical method with low cost and high speed.

Disposable luminol copolymer-based biosensor for uric acid in urine.

A new electrochemiluminescent (ECL) disposable biosensor for uric acid was manufactured by immobilization in a double-layer design of luminol as a copolymer with 3,3',5,5'-tetramethylbenzidine (TMB) and the enzyme uricase in chitosan on gold screen-printed cells. The good mechanical and improved electroluminescent characteristics of the new copolymer poly(luminol-TMB) make it possible to determine uric acid by measuring the growing ECL emission with the analyte concentration. The combination of enzymatic selectivity with ECL sensitivity results in a disposable analytical device with a linear range for uric acid from 1.5×10(-6) to 1.0×10(-4) M, a limit of detection of 4.4×10(-7) M and a precision of 13.1% (1.0×10(-5) M, n=10) as relative standard deviation. Satisfactory results were obtained for uric acid determination in 24h-urine samples compared to a reference procedure. This uric acid biosensor can be used as a low-cost alternative to conventional methods.

Copolymerization of luminol on screen-printed cells for single-use electrochemiluminescent sensors.

An efficient electrochemiluminescent (ECL) single-use sensor for H(2)O(2) is presented based on an electropolymerized film prepared on screen-printed gold electrode (gold SPE). A study of the copolymerization of luminol in the presence of different monomers was carried out. The polymeric films were grown potentiodynamically with a potential interval between -0.2 and 1.0 V in 0.2 M H(2)SO(4) and were characterized by their electrochemical, electrochemiluminescent, and superficial features. The polymer with the most efficient growth and ECL emission was poly(luminol-3,3',5,5'-tetramethylbenzidine) at 1:5 ratio. These prepared SPE cells present good mechanical and photoemissive properties. A semi-logarithmic linearization shows a noticeable four decade-width concentration range with a limit of detection (LOD) of 2.6 × 10(-9) M and a precision of 10.2% (n = 5; as relative standard deviation, RSD) in the medium range level. The described SPE ECL sensors will be useful for the determination of oxidase substrates in ECL single-use biosensors.

Disposable electrochemiluminescent biosensor for lactate determination in saliva.

An electrochemiluminescence-based disposable biosensor for lactate is characterized. The lactate recognition system is based on lactate oxidase (LOx) and the transduction system consists of luminol. All the needed reagents, luminol, LOx, BSA, electrolyte and buffer have been immobilized by a Methocel membrane placed on the working electrode of the screen-printed electrochemical cell. The measurement of the electrochemiluminescence (ECL) is made possible via a photocounting head when 50 microl of sample is placed into the screen-printed cell with a circular container containing the disposable sensing membrane. The compositions of the membrane and reaction conditions have been optimized to obtain adequate sensitivity. The disposable biosensor responds to lactate after 20 s when two 1 s pulses at 0.5 V are applied to obtain the analytical parameter, the ECL initial rate. The linearized double logarithmic dependence for lactate shows a dynamic range from 10(-5) to 5 x 10(-4) M with a detection limit of 5 x 10(-6) M and a sensor-to-sensor repeatability, as relative standard deviation, RSD, of 3.30% at the medium level of the range. The ECL disposable biosensor was applied to the analysis of lactate in human saliva as an alternative procedure for obtaining the lactate level in a non-invasive way. Interferences coming from components of saliva were studied and eliminated in a simple way that was easy to handle. The procedure was validated for use in human saliva, comparing the results against an enzymatic reference procedure. The proposed method is quick, inexpensive, selective and sensitive and uses conventional ECL instrumentation.

One-shot lactate chemiluminescent biosensor.

A new chemiluminescence-based one-shot biosensor for lactate is described. The lactate recognition system is based on lactate oxidase (LOx) and the transduction system consists of luminol, peroxidase from Arthromyces ramosus (ARP) and metallic aluminum, all immobilized in a polyion complex membrane. The measurement of the chemiluminescence in a luminometer when 1 mL of sample is injected into a conventional cell containing the disposable sensing membrane makes it possible to determine lactate. The compositions of the membrane and reaction conditions have been optimized to obtain adequate sensitivity. The one-shot biosensor responds to lactate rapidly, with the typical CL acquisition time being 2 min, with a linearized logarithmic dependence whose dynamic range was from 5 x 10(-5) to 4 x 10(-3) with a detection limit of 9.2 x 10(-6)M and a sensor-to-sensor reproducibility (relative standard deviation, R.S.D.) of 5.5% at the medium level of the range. The performance of the chemiluminescent one-shot biosensor was tested for the analysis of lactate in yoghurt, validating the results against an enzymatic reference procedure. The proposed method is quick, inexpensive and sensitive and uses conventional CL instrumentation.

Chemiluminescence determination of sodium 2-mercaptoethane sulfonate by flow injection analysis using cerium(IV) sensitized by quinine.

A flow injection method with chemiluminescence (CL) detection is proposed for the determination of sodium 2-mercaptoethane sulfonate (MESNA), a drug often used to reduce the urotoxic effects of antineoplastic alkylating agents. The procedure is based on the reaction of the thiol with Ce(IV) in a sulfuric acid medium and measurement of the CL intensity produced by quinine used as a sensitizer. The optimum conditions for CL emission were investigated. Using the CL peak height as the analytical parameter, MESNA was determined over the concentration range 0.29-2.21 ng (1.97-9.85 mug.l(-1)) with a detection limit of 0.21 ng (1.38 mug.l(-1)) and a relative standard deviation (R.S.D.) of 4.1%. The method was applied satisfactorily to the determination of MESNA in pharmaceutical preparations with percentages of recovery between 94 and 105.