Oxalyldiimidazole Chemiluminescence Research Articles

A highly selective and sensitive competitive aptasensor capable of quantifying thrombin elevated with side effects in severe COVID-19

To protect critical injury from blood clots with side effects in severe COVID-19, a highly selective and sensitive biosensor was developed for the quantification of trace levels of thrombin using the combination of a DNA aptamer (TBA) of thrombin and a complementary strand of TBA. TBA rapidly binds with thrombin, whereas it slowly binds with the complementary strand to form a double stranded DNA (dsDNA). SFC green intercalated into dsDNA cannot emit light in 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) reaction because high-energy intermediates formed from ODI-CL reaction cannot transfer energy to SFC trapped in dsDNA. However, SFC freely existing with the formation of G-quadruplex from the reaction of thrombin and TBA emits bright chemiluminescence because the high-energy intermediates can transfer energy to SFC (or camel) in solution. Thus, the brightness of light emitted in ODI-CL reaction was proportionally enhanced with the increase of thrombin in a sample due to the increase of G-quadruplex and reduction of dsDNA. The limit of detection (LOD) of the label free aptasensor operated with good linear calibration curve (10–320 mU/ml) was as low as 3 mU/ml (or 43 pM). Also, the biosensor was quantified trace levels of thrombin with good accuracy, precision, and reliability.

A cost-effective and rapid aptasensor with chemiluminescence detection for the early diagnosis of prostate cancer

A rapid aptasensor capable of early diagnosing prostate cancer was developed using a dual aptamer, which is composed of a prostate specific antigen (PSA) aptamer and hemin aptamer connected by a linker composed of 5 adenines. The dual aptamers immobilized on the surface of paramagnetic beads were able to competitively bind with PSA or hemin in human serum for 20 min at room temperature. After the incubation, the dual aptamers were separated from human serum using a magnetic separator. After the procedure, the mixture of Amplex Red and H2O2 in Tris–HCl (pH 8) was added in a microcentrifuge tube containing the dual aptamers. Resorufin was formed from the reaction of Amplex Red and H2O2 in the presence of horseradish peroxidase (HRP) mimicking DNAzyme formed from the reaction of hemin and the dual aptamer. The concentration of resorufin formed from the reaction was proportional to the concentration of HRP mimicking DNAzyme. Resorufin emitted bright red light with the addition of 1,1′-oxalyldiimidazole (ODI) chemiluminescence reagents such as ODI and H2O2. The brightness of the emission light was proportionally reduced with the increase of PSA in human serum due to the reduction of DNAzyme concentration with the binding of PSA and PSA aptamer linked to hemin aptamer. Using the calibration curve of the new aptasensor, trace levels of PSA in human serum was rapidly quantified for the early diagnosis of prostate cancer. The limit of detection (LOD = 3 s/slope) of the aptasensor was as low as 0.1 ng/ml. The accuracy, precision, and recovery of the aptasensor were studied with statistically acceptable methods.

Cost-effective monitoring of microRNA-205 applied as a biomarker using G-quadruplex DNAzyme and 1,1’-oxalyldiimidazole chemiluminescence

Trace levels of microRNA-205, known as a biomarker of lung cancer, in human serum was quantified for the first-time using G-quadruplex DNAzyme linked to detection complementary probe and 1,1’-oxalyldiimidazole chemiluminescence (ODI-CL). First, capture complementary probes immobilized on the surface of paramagnetic bead selectively bound with microRNA-205 existing in human serum. Then, with the addition of detection complementary probe linked to hemin aptamer, a complex linked to hemin aptamer was formed with the completion of hybridization between microRNA-205 and two complementary probes. With the addition of hemin in the solution, finally, a complex linked to G-quadruplex DNAzyme was formed from the interaction of hemin aptamer and hemin. Resorufin, luminescent dye, was formed from the reaction of Amplex Red and H2O2 in the presence of the complex linked to DNAzyme acting as a horseradish peroxidase (HRP)-mimicking enzyme. The concentration of resorufin formed from the reaction was dependent on the concentration of microRNA-205 in human serum. Thus, the brightness of resorufin emitted in ODI-CL reaction was enhanced with the increase of microRNA-205. The limit of detection (LOD) of the biosensor with ODI-CL detection, capable of sensing microRNA-205 (dynamic range: 0.4–62.5 nM), was as low as 0.13 nM. It was confirmed that the biosensor can quantify trace levels of microRNA-205 with statistically acceptable accuracy, precision, and recovery.

Highly sensitive chemiluminescence enzyme immunoassay for the quantification of carcinoembryonic antigen in the presence of an enhancer and a stabilizer

Using the advantages of phenol red, a signal enhancer, and bovine serum albumin (BSA), a stabilizer of horseradish peroxidase (HRP), added in HRP enzyme reaction of Amplex Red and H2O2, highly sensitive 1,1′-oxalyldiimidazole chemiluminescence enzyme immunoassay (ODI-CLEIA) was developed to rapidly quantify trace levels of carcinoembryonic antigen (CEA) in human serum. Phenol red acts as an enhancer in ODI-CLEIA while BSA supported rapid and stable activation of HRP. The CL emission of resorufin formed from the HRP enzyme reaction in the presence of BSA and phenol red was about 70-fold brighter than that in the absence of both materials. ODI-CLEIA in the presence of BSA (1.5 mg/ml).and phenol red (1 mM) was able to rapidly analyze CEA in human serum with the wide linear calibration curve (2.5–100 ng/ml). The limit of detection (LOD = 3σ/slope) of ODI-CLEIA was as low as 0.19 ng/ml. Additionally, it was confirmed that the accuracy, precision, and reproducibility of ODI-CLEIA in the presence of BSA and phenol red were good with the statistically acceptable error range.

Chemiluminescent dual-enzyme immunoassays capable of simultaneously quantifying carbohydrate antigen 19–9 and carcinoma embryonic antigen in a sample

Using the internal chemiluminescence resonance energy transfer (Inter-CRET) of luminescent dye and the high-energy intermediate formed in 1,1‘-oxalyldiimidazole chemiluminescence (ODI-CL) reaction, we developed for the first time dual-enzyme immunoassays operated with alkaline phosphatase (ALP) and horseradish peroxidase (HRP) for the simultaneous quantifications of carbohydrate antigen 19–9 (CA19-9) and carcinoma embryonic antigen (CEA) in a sample. Fluorescein formed from the ALP enzyme immunoassay emits green light in ODI-CL reaction, while resorufin formed from the HRP enzyme immunoassay emits red light. Green and red CL lights emitted in a detection cell were measured individually with two photomultiplier tubes with an optical filter capable of sensing green or red emission without interferences. The limits of detection for CA 19–9 (0.09 U/ml) and CEA (0.11 ng/ml) determined using the accurate and reproducible dual-enzyme immunoassays were as low as those calculated using the conventional single-enzyme immunoassays capable of sensing CA 19–9 or CEA in a sample. In conclusion, we confirmed that the highly selective dual enzyme immunoassays can be applied as a new analytical method capable of early diagnosing and monitoring colon and pancreatic cancers with the simultaneous analyses of CA 19–9 and CEA.

Self-enzyme chemiluminescence immunoassay capable of rapidly diagnosing the infection of influenza A (H1N1) virus

A highly sensitive self-enzyme immunoassay with 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) detection was developed for the first time to quantify influenza A (H1N1) virus. A specific capture antibody, capable of capturing hemagglutinin (HA) subtypes of H1N1, was immobilized on the surface of the magnetic Au-Fe3O4 nanocomposite. Neuraminidase (NA) subtype of H1N1 was acts as an enzyme in the self-enzyme immunoassay. A sample mixed with HA antibodies immobilized on the surface of magnetic Au-Fe3O4 nanocomposites was incubated for 1 h at 37 °C. After the incubation, magnetic Au-Fe3O4 nanocomposites separated with a magnetic bar were washed 3 times using phosphate buffered saline with Tween-20 (PBST). Then, 4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid (MUNANA), a fluorogenic substrate of NA, was added and incubated for 10 min to produce 4-Methylumbelliferone (4-MU) from the enzyme reaction between MUNANA and NA of H1N1. After the incubation, the solution containing 4-MU emitted bright light with the addition of ODI-CL reagents (e.g., H2O2 and ODI). The relative CL intensity of 4-MU was proportionally enhanced with the increase of H1N1. Also, the brightness and color of 4-MU light emitted from the self-enzyme immunoassay was dependent on pH. The self-enzyme immunoassay was able to analyze trace levels of influenza A (H1N1) virus with good accuracy, precision, reproducibility and excellent selectivity. The limit of detection (LOD = 3σ/slope) was as low as 0.19 U/ml. We expect that the self-enzyme immunoassay can be applied as a cost-effective and rapid method capable of selectively quantifying a specific influenza A virus such as H1N1, H3N2, and H5N1.

Enzyme-free chemiluminescence immunoassay for the determination of thyroid stimulating hormone

Based on the chemiluminescent resonance energy transfer (CRET) from high-energy intermediate formed in 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) reaction to fluorescent microsphere-conjugated detection antibody, a cost-effective and highly sensitive enzyme-free sandwich immunoassay with chemiluminescence detection was developed for the early diagnosis of thyroid cancer. Thyroid stimulating hormone (TSH) primary antibody immobilized on paramagnetic bead captures rapidly TSH in human serum within 15min. Also, TSH detection antibody-conjugated yellow-green microsphere, capable of emitting green light, rapidly interacted with TSH-bound TSH primary antibody immobilized on paramagnetic bead during the 30-min incubation at room temperature. The sandwich complexes immobilized on paramagnetic in PBS buffer (pH 7.4) emit bright green light with the addition of ODI-CL reagents (e.g., H2O2, ODI). The dynamic range of enzyme-free sandwich immunoassay was 0.037–18μIUml−1 with 3–5% coefficient of variation. The limit of detection (LOD=background+3σ) of the enzyme free sandwich immunoassay was as low as 0.011μIUml−1. We expect that the enzyme free sandwich immunoassay with ODI-CL detection can be a new assay method for the early diagnosis of thyroid cancer with statistically acceptable accuracy, precision, and reproducibility.

All-in-one dual-aptasensor capable of rapidly quantifying carcinoembryonic antigen

Using a dual DNA aptamer (CEA aptamer linked to hemin aptamer), capable of rapidly capturing carcinoembryonic antigen (CEA) and hemin, an all-in-one dual-aptasensor with 1,1′-oxalyldiimidazole (ODI) chemiluminescence detection was developed for the early diagnosis of human cancer. CEA and hemin competitively bound with the dual DNA aptamer while the mixture in a detection cell was incubated for 30min at room temperature. When Amplex Red and H2O2 were added in the detection cell after the incubation, the yield of resorufin formed from the reaction Amplex Red and H2O2 depended on the concentration of HRP-mimicking G-quardruplex DNAzyme formed from the binding interaction between hemin and the dual DNA aptamer. Bright red light was observed with the addition of ODI and H2O2 in the detection cell containing resorufin. Relative CL intensity of all-in-one dual-aptasensor, operated with the competitive reaction of CEA and hemin in the presence of the dual aptamer, was exponentially decreased with the increase of CEA concentration in human serum. The limit of detection (LOD=3σ) of the all-in-one dual-aptasensor which operated with excellent accuracy, precision, and reproducibility was as low as 0.58ng/ml. The good correlation between the easy to use all-in-one dual-aptasensor and conventional enzyme-linked immunosorbent assay (ELISA), operated with time consuming procedures (e.g., long incubations and multiple washings), indicates that the rapid all-in-one dual-aptasensor can be applied as a novel clinical tool for the early diagnosis of breast cancer.

Development of 1,1′-oxalyldiimidazole chemiluminescent biosensor using the combination of graphene oxide and hairpin aptamer and its application

Highly sensitive biosensor with 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) detection was developed to rapidly quantify Vibrio (V) parahaemolyticus without time-consuming procedures such as multiple long-incubations and washings. When V. parahaemolyticus in Tris-HCl (pH 7) and hairpin DNA aptamer conjugated with TEX615 in DNA free deionized water were consecutively added in PBS buffer (pH 7.4) containing graphene oxides (GOs), V. parahaemolyticus and GOs bind competitively to hairpin DNA aptamer conjugated with TEX615 during 10 min of incubation at room temperature. Brightness of light immediately emitted with the addition of ODI-CL reagents (e.g., ODI, H2O2) after the incubation was dependent on the concentration of V. parahaemolyticus in a sample. The dynamic range of linear calibration curve for the quantification of V. parahaemolyticus in a sample was from 4375 to 70,000 cells/ml. The limit of detection (LOD = background + 3 × standard deviation, 2230 cells/ml) of the biosensor operated with good accuracy, precision, and recovery was lower than those of conventional assay methods such as time-consuming and expensive enzyme-linked immunosorbent assays.

Role of background observed in aptasensor with chemiluminescence detection

One-step chemiluminescent aptasensor was developed using chemically initiated electron exchange luminescence (CIEEL) between high-energy intermediate formed from 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) reaction and G-quadruplex (ochratoxin A (OTA)-bound aptamer conjugated with TEX615) generated. The sensitivity of chemiluminescent aptasensor, optimized with various variables (e.g., property of microfibers fabricated with 3,4,9,10-perylenetetracarboxylic dimide, determination of fluorescent dye labeled with aptamer, physical properties of buffer solution), was dependent on the background (concentration of high-energy intermediate) generated in ODI-CL reaction. The limit of detection (LOD=background+3×standard deviation, 0.5nM) of ODI-CL aptasensor with lower background was lower than that (3.7nM) with 20 times higher background. Also, the ratio of signal to background (S/B) of ODI-CL aptasensor with low background was about 5-fold higher than that with high background. The sensitivities of ODI-CL aptasensors, with low as well as high background, capable of accurately and precisely quantifying OTA within 10min, were better than those of fluorescent aptasensors and as good as those of highly sensitive but time-consuming competitive enzyme-linked immune-sorbent assays (ELISAs) using expensive antibody produced with the sacrifice of small animals.

Rapid hybridization using graphene oxide and 1,1′-oxalyldiimidazole chemiluminescence

A rapid hybridization method using graphene oxide and highly sensitive 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) detection was developed from an understanding of the chemical and physical interactions between single strand DNA (ssDNA) molecules and nanoparticles (e.g., multi-walled carbon nanotubes, graphene oxide, gold and silver nanoparticles). The efficiency of hybridization between mutated ssDNA and a complementary probe conjugated with TEX615 was dependent on four different variables: pH, temperature, incubation time, and properties of nanoparticles capable of capturing excess complementary probes remaining after the hybridization. A critical problem observed when mutated ssDNAs rapidly bound with complementary probe-conjugated TEX615 was that three different types of possible mismatched ssDNAs also slowly and competitively hybridized with complementary probe-conjugated TEX615. The problem was solved upon addition of three different types of complementary probes, not conjugated with TEX615, in the solution because mismatched DNAs hybridize with their complementary probes while mutated ssDNAs bind with complementary probe-conjugated TEX615. The new hybridization method using graphene oxide and ODI-CL detection quantified trace levels of mutated ssDNAs in a sample containing mismatched ssDNAs within 15 minutes without any interference from mismatched ssDNAs.

Role of magnetic Fe3O4 graphene oxide in chemiluminescent aptasensors capable of sensing tumor markers in human serum

Using π–π stacking interaction between magnetic Fe3O4 graphene oxide nanoparticles and impurities (e.g., biomolecules, free prostate specific antigen (PSA) aptamer-conjugated TEX615 that does not bind with PSA) in human serum, a rapid aptasensor with 1,1′-oxalyldiimidazole chemiluminescence (ODI-CL) detection was developed for the quantification of PSA, a tumor marker. The complexes, PSA bound with PSA aptamer-conjugated TEX615, were formed in human serum while the mixture (1 : 1 volume ratio) of PSA in 25% human serum diluted in Tris–HCl buffer (pH 7.5) and PSA aptamer-conjugated TEX615 in Tris–HCl buffer was incubated for 30 minutes. After the incubation, the impurities capable of reducing the efficiency of the aptasensor were removed using magnetic Fe3O4 graphene oxide nanoparticles. With the addition of ODI CL reagents (e.g., ODI, H2O2), finally, PSA in each sample was quantified within 1 second. The sensitivity of the aptasensor with good accuracy, precision, and recovery was as good as those of enzyme immunoassays with absorbance and chemiluminescence detections operated using time-consuming procedures such as multiple-time incubation and washing and expensive and intractable antibodies.

Roles of CdS quantum dots in 1,1′-oxalyldiimidazole chemiluminescence

The roles of CdS nanocrystals (quantum dots) stabilized by oleic acid ligands in toluene were investigated in the 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) reaction generated under various environmental conditions. CdS excited by high-energy intermediates formed in ODI-CL reaction emitted dim CL. Also, CdS acted as a catalyst to enhance the yield of high-energy intermediates, capable of transferring energy to fluorescent molecules, in aqueous ODI-CL reactions, whereas it acted as a strong quencher, capable of inhibiting the CL emission of excited fluorescent molecules, in non-aqueous ODI-CL reactions. Based on the role of CdS in the aqueous ODI-CL reaction, the limit of detection (LOD = signal/noise = 3, 0.1 μM) determined to quantify glucose using aqueous ODI-CL reaction in the presence of 2.75 μM CdS was four times lower than that in the absence of CdS. The range of recovery determined in the aqueous ODI-CL reaction in the presence of CdS was 91.7-104%. We expect that the aqueous ODI-CL reaction in the presence of CdS can be applied as a highly sensitive sensor in various research fields such as bioanalytical chemistry, environmental engineering, homeland security, and toxicology.

1,1′-Oxalyldiimidazole chemiluminescent enzyme immunoassay capable of simultaneously sensing multiple markers

In order to rapidly and simultaneously quantify and screen trace levels of multiple biomarkers in a single sample, rapid 1,1′-oxalyldiimidazole chemiluminescence (ODI CL) was applied as a biosensor of immunoassays using various enzymes such as alkaline phosphatase (ALP) and horseradish peroxidise (HRP). (1) Fluorescein was formed from the reaction of fluorescein diphosphate (FDP) and immuno-complex conjugated with ALP. (2) Resorufin was formed from the reaction between Amplex Red and H 2O 2 in the presence of immuno-complex conjugated with HRP. When ODI CL reagents (H 2O 2 in isopropyl alcohol, ODI in ethyl acetate) were injected in a test tube or strip-well containing fluorescein and resorufin formed from above two reactions a bright CL emission spectrum having two peaks (518 nm for fluorescein and 602 nm for resorufin) was observed. The two peaks can be independently quantified with an appropriate statistical tool capable of deconvoluting multiple emission peaks. In conclusion, we expect that ODI chemiluminescent enzyme immunoassays (CLEIAs) using a couple of enzymes conjugated with antigen or antibody and substrates can rapidly and simultaneously quantify and screen multiple biomarkers in a single sample.

Rapid monitoring of alkaline phosphatase in raw milk using chemiluminescence detection.

A simple biosensor with 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) detection capable of rapidly quantifying and screening alkaline phosphatase (ALP) in raw and pasteurized milk was developed as an indicator for confirming whether commercial milk is properly pasteurized. Fluorescein was formed when standards containing 1.0% milk with different activities of ALP and samples containing 1.0% raw milk were incubated with fluorescein diphosphate (FDP) for 15 min at room temperature. The relative CL intensity of fluorescein measured with the addition of 80 mM H2O2 and ODI formed from the reaction of 2.0 μM bis(2,4,6-trichlorophenyl) oxalate and 10.0 μM 4-methyl imidazole in ethyl acetate was proportional to the concentration of ALP in milk. The range (39∼2500 mU/L) of linear calibration curve (R2 = 0.998) for the quantification of ALP in milk using ODI-CL detection was wider than those using currently applied fluorescence and 1,2-dioxetane CL detections. Also, the limit of detection (3.7 mU/L) determined using the former detection, which has good precision, was lower than those reported using the latter detections. In conclusion, the cost-effective and highly sensitive biosensor with ODI-CL detection can be applied to monitor whether milk is pasteurized according to acceptable ALP activities threshold level (350 mU/L) for public safety newly adopted by US and EU and the internal investigation level (100 mU/L) proposed by EU.

Fieldable flow injection analysis system with chemiluminescence detection capable of quantifying.

We developed a highly sensitive biosensor for quantifying acetylcholine (ACh) using flow injection analysis system with 1,1'-oxalyldiimidazole chemiluminescence (ODI-CL) detection designed based on the principle of liquid core waveguide. ACh in Tris-HCl (pH 8.5) was incubated with the mixture of 1.0 U/ml acetylcholinesterase, 0.5 U/ml choline oxidase, 0.04 U/ml horseradish peroxidase, and 1.0 μM Amplex Red in PBS (pH 7.4) for 15 min at room temperature. The concentration of resorufin formed from the consecutive enzyme reactions was proportional to the concentration of ACh in analytical sample. The dynamic range of linear calibration curve (y = 12444x + 11617, R2 = 0.998) for the quantification of ACh using the biosensor with ODI CL detection was 0.7∼11.3 μM. The limit of detection (LOD = background noise + 3σ) of the biosensor was as low as 0.14 μM. Based on the results of recovery test and linearity study, finally, we confirmed that FIA system with ODI CL detection is accurate, precise, and reproducible.

Advent of innovative chemiluminescent enzyme immunoassay

Using 1,1′-oxalyldiimidazole (ODI) chemiluminescence detection, a new chemiluminescent enzyme immunoassay (CLEIA) was developed to quantify prostate specific antigen (PSA) in human serum. The results observed in ODI CLEIA were compared with those obtained in commercially available enzyme linked immunosorbent assay (ELISA), fluorescence enzyme immunoassay (FEIA), and luminol CLEIA. PSA complex-conjugated horseradish peroxidase (HRP) was formed from one-step sandwich immunoreaction of PSA, PSA primary antibody and PSA secondary antibody-conjugated HRP for 15 min in a strip-well at 36.5 °C. CL substrate solution (Amplex Red and H 2O 2 in PBS buffer, pH 7.4) was added in the washed strip-well and incubated for 10 min at room temperature. When resorufin formed in this process was mixed with 1,1 ′-oxalyldi-4-methylimidazole and H 2O 2 in a testing tube, rapid and bright CL was observed. Detection limit (0.035 ng/ml) of PSA in ODI CLEIA was much lower than those (0.50 and 0.25 ng/ml) in commercially available ELISA and luminol CLEIA even though total incubation time of the former (25 min) was shorter than those of the latter (45 and 35 min). Also, the dynamic range (0–100 ng/ml, R 2 = 0.9996) of ODI CLEIA was wider than those of other EIAs. In conclusion, the excellent correlation ( r = 0.9767) between ODI CLEIA and Advia Centaur XP Immunoassay System indicates that the accurate, precise, and rapid ODI CLEIA can be applied as a novel CLEIA capable of diagnosing and monitoring various diseases.

Solvent and pH effects on fast and ultrasensitive 1,1'-oxalyldi(4-methyl)imidazole chemiluminescence.

Solvent and pH effects on fast and ultrasensitive 1,1'-oxalyldi(4-methyl)imidazole chemiluminescence (OD4MI-CL) were studied. The influences of these two factors on the complex OD4MI-CL reaction are discussed within a conceptual prototype for developing aqueous and non-aqueous capillary electrophoresis (ACE and NACE) devices with OD4MI-CL detection. The reaction channel length and OD4MI yield from the reaction between bis(2,4,6-trichlorophenyl) oxalate (TCPO) and 4-methylimidazole in the channel will be influenced by pH, water volume fraction, and cosolvent properties of the solution. Optimum OD4MI-CL efficiency is observed at pH 6.5 when 1-propanol, which has a low dielectric constant (epsilon = 20.8), is used as the NACE solvent in the separation channel. Water (epsilon = 80.1), the solvent in the ACE separation channel, acts similarly to a high dielectric constant organic solvent in NACE because the disadvantages normally associated with TCPO-CL reactions in water disappear due to the faster OD4MI-CL reaction versus OD4MI decomposition in aqueous solution. Therefore, it is expected that the OD4MI-CL detection system can be used in both NACE and ACE devices without requiring detector modifications. We also conclude that OD4MI-CL detection in NACE and ACE devices will be much more sensitive than the TCPO-CL detection used in current NACE devices.

Peroxyoxalate chemiluminescence in aqueous solutions: Coupling of immobilized enzyme reactors and 1,1′-oxalyldiimidazole chemiluminescence reaction to flow-injection analysis and liquid chromatographic systems

A highly sensitive method for the determination of enzymatically generated hydrogen peroxide in flow-injection analysis (FIA) and liquid chromatography (LC) has been developed. A dual-line flow system is used, one carrier (or eluent) delivering the analyte and the other one the chemiluminescent reagent 1,1′-oxalyldiimidazole (ODI). The results show that the composition of the analyte flow line is not critical for the chemiluminescence detection step; even purely aqueous buffers, as generally applied if immobilized enzyme reactors (IMERs) are involved in FIA and LC, can be used without loss of sensitivity. IMERs containing either glucose oxidase or acetylcholine esterase/choline oxidase were incorporated in this flow line and favourable detection limits (S/N = 3) were obtained, i.e. 3 nM for glucose and 50 nM for acetylcholine and choline. The performance of the approach in real-sample analysis was tested by determining glucose and choline in urine samples.