Factors influencing level of hydrogen peroxide in exhaled breath condensate

Hydrogen peroxide (H₂O₂) in exhaled breath condensate (EBC) has been proposed as a marker for oxidative stress in the airways. The aim of the present study was to evaluate the measurement of H₂O₂ in EBC with or without use of a nose clip, and the influence of mouth rinsing, sampling time and storage.An elevated H₂O₂ level was seen during nasal breathing compared to mouth breathing with nose clip (3.4 pmol/s vs. 2.1 pmol/s, p = 0.02). Breathing through the mouth, using a nose clip, was therefore practiced in all experiments. The H₂O₂ levels were increased when mouth rinsing was performed using an acid buffer (1.4 pmol/s vs. 1.9 pmol/s, p = 0.03). 15 min sampling time decreased the H₂O₂ output by almost 50% compared with 2 min sampling time (1.2 vs. 0.6 pmol/s, p = 0.03). When samples were left unattended for 15 min no change in H₂O₂ concentration in the EBC was seen.We found no significant differences in H₂O₂ levels between samples stored for 4 weeks at − 80 °C and samples analysed directly; however, a significant decrease in the levels was seen for samples stored for 4 weeks at − 20 °C.In conclusion, the method of EBC collection and storage plays an important role in reducing variability within and between individuals.     

Fast determination of paeonol in plasma by headspace solid-phase microextraction followed by gas chromatography-mass spectrometry

Paeonol is the active component in the traditional Chinese medicines (TCMs), such as Cynanchum paniculatum, which has been used to treat many diseases, such as eczema. In this work, a simple, rapid and sensitive method was developed for the determination of paeonol in rabbit plasma, which was based on headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS). The extraction parameters of fiber coating, sample temperature, extraction time, stirring rate and ion strength were systemically optimized; the method linearity, detection limit and precision were also investigated. It was shown that the proposed method provided a good linearity (0.02-20 μg mL⁻¹, R² > 0.990), low detection limit (2.0 ng mL⁻¹) and good precision (R.S.D. value less than 8%). Finally, GC/MS following HS-SPME was applied to fast determination of paeonol in rabbit plasma at different time point after oral demonstration of Cynanchum paniculatum essential oil. The experimental results suggest that the proposed method provided an alternative and novel approach to the pharmacokinetics study of paeonol in the TCMs.     

Chiral analyses of dextromethorphan/levomethorphan and their metabolites in rat and human samples using LC-MS/MS

In order to develop an analytical method for the discrimination of dextromethorphan (an antitussive medicine) from its enantiomer, levomethorphan (a narcotic) in biological samples, chiral analyses of these drugs and their O-demethyl and/or N-demethyl metabolites in rat plasma, urine, and hair were carried out using LC-MS/MS. After the i.p. administration of dextromethorphan or levomethorphan to pigmented hairy male DA rats (5 mg/kg/day, 10 days), the parent compounds and their three metabolites in plasma, urine and hair were determined using LC-MS/MS. Complete chiral separation was achieved in 12 min on a Chiral CD-Ph column in 0.1% formic acid–acetonitrile by a linear gradient program. Most of the metabolites were detected as being the corresponding O-demethyl and N, O-didemethyl metabolites in the rat plasma and urine after the hydrolysis of O-glucuronides, although obvious differences in the amounts of these metabolites were found between the dextro and levo forms. No racemation was observed through O- and/or N-demethylation. In the rat hair samples collected 4 weeks after the first administration, those differences were more clearly detected and the concentrations of the parent compounds, their O-demethyl, N-demethyl, and N, O-didemethyl metabolites were 63.4, 2.7, 25.1, and 0.7 ng/mg for the dextro forms and 24.5, 24.6, 2.6, and 0.5 ng/mg for the levo forms, respectively. In order to fully investigate the differences of their metabolic properties between dextromethorphan and levomethorphan, DA rat and human liver microsomes were studied. The results suggested that there might be an enantioselective metabolism of levomethorphan, especially with regard to the O-demethylation, not only in DA rat but human liver microsomes as well. The proposed chiral analyses might be applied to human samples and could be useful for discriminating dextromethorphan use from levomethorphan use in the field of forensic toxicology, although further studies should be carried out using authentic human samples.     

Liquid chromatographic/electrospray mass spectrometric determination (LC/ESI-MS) of chelerythrine and dihydrochelerythrine in near-critical CO2 extracts from real and spiked plasma samples

Two polar benzo[c]phenanthridine alkaloids, chelerythrine (CHE) and dihydrochelerythrine (DHCHE), were extracted at 35 °C and 10 MPa (15 MPa for real samples) from real and spiked plasma samples with acceptable recoveries (95.1% and 81.0%, respectively) using near-critical CO₂ modified with aqueous (1:1, v/v) methanol. The alkaloids were quantified by a liquid chromatographic/electrospray mass spectrometric (LC/ESI-MS) method on a Zorbax SB-CN column (75 mm × 4.6 mm, 3.5 μm particle size) using methanol (organic phase) and 50 mM ammonium formiate (aqueous phase) as a mobile phase. A linear gradient 0-1 min, isocratic at 60% organic phase (v/v); from 1.0 to 7.0 min, 60-71% organic phase (v/v); from 7.0 to 18.0 min, 71-60% organic phase (v/v) was applied. The limit of detection was 1.22 ng (3.50 pmol) for CHE and 0.95 ng (2.72 pmol) for DHCHE per 1 ml of the sample. The linearity of the calibration curves was satisfactory as indicated by coefficients of determination 0.9979 and 0.9995 for CHE and DHCHE, respectively. Repeatability and intermediate precision (average R.S.D.s) were 1.0-1.5%, accuracy was in the range 99.7-100.3%. Average recovery was 100.1% for both, standard solutions and spiked plasma extracts. Three samples of real rat plasma were extracted and analysed to test the method.     

Formation of γ-Fe2O3 nanoparticles and vacancy ordering: An in situ X-ray powder diffraction study

The formation of maghemite, γ-Fe₂O₃ nanoparticles has been studied by in situ X-ray powder diffraction. The maghemite was formed by thermal decomposition of an amorphous precursor compound made by reacting lauric acid, CH₃(CH₂)₁₀COOH with Fe(NO₃)₃·9H₂O. It has been shown that cubic γ-Fe₂O₃ was formed directly from the amorphous precursor and that vacancy ordering starts about 45 min later at 305 °C resulting in a tripled unit cell along the c-axis. The kinetics of grain growth was found to obey a power law with growth exponents n equal to 0.136(6) and 0.103(5) at 305 and 340 °C, respectively. Particles with average sizes of 12 and 13 nm were obtained in 86 and 76 min at 305 and 340 °C, respectively. The structure of cubic and vacancy ordered phases of γ-Fe₂O₃ was studied at 305 °C by Rietveld refinements.     

Determination of kynurenine levels in rat plasma by high-performance liquid chromatography with pre-column fluorescence derivatization

Kynurenine (KYN), a tryptophan metabolite, is a crucial compound for modulating neurotransmission because it can be metabolized in vivo into both quinolinic acid and kynurenic acid, which are the agonist and antagonist, respectively, of N-methyl-d-aspartate receptor. For the highly sensitive detection of KYN by high-performance liquid chromatography (HPLC), a fluorescence derivatization of KYN with a benzofurazan-type fluorogenic reagent, 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was investigated in the present study. KYN was derivatized with DBD-F (DBD-KYN) at 60 °C for 30 min, and separated on an octadecylsilica column with a gradient elution of the mobile phase, which consists of 0.1% formic acid in acetonitrile/methanol/water. DBD-KYN was detected fluorimetrically at 553 nm with an excitation wavelength of 431 nm. The limits of detection and quantification were approximately 0.30 pmol [signal-to-noise ratio (S/N) 3] and 1.0 pmol (S/N, 10) on column, respectively. Plasma KYN levels were successfully determined using 10 μL of rat plasma with satisfactory precision and accuracy. Intra- and inter-day precisions and accuracies were 1.7-6.8%, and −10 to 9.6%, respectively. KYN levels in plasma of male Sprague-Dawley rats (7 weeks old) were approximately 2.4 ± 0.32 μmol L⁻¹ (n = 4). The proposed HPLC method was applied to determine KYN levels in the plasma of ketamine-treated rats—the animal model of schizophrenia.     

Isolation of the retinal isomers from the isomerization of all-trans-retinal by flash countercurrent chromatography

The isolation of the retinal isomers from all-trans-retinal was performed by flash countercurrent chromatography. In each separation, isomerization reaction solution of 200 mg all-trans-retinal could be loaded on a 1200 mL of high-speed countercurrent chromatographic column with 5 mm bore, eluted by a mobile phase flow rate of 25 mL/min, resulting in 63 mg of 11-cis-retinal, 24 mg of 13-cis-retinal and 26 mg of 9-cis-retinal with purities more than 95%. n-Hexane–acetonitrile (3:1) was used as the solvent system which possesses the advantages of simplicity, re-use of the solvent and multiple injections. This method could be used to prepare 13-cis-retinal, 11-cis-retinal and 9-cis-retinal for the photoisomerization investigation, such as the effect of 11-cis-retinal in the visual system.     

Immunochromatographic flow-injection method for detection of human serum immunoglobulin G antibodies to Helicobacter pylori

This study reports on the development of an immunochromatographic flow-injection (FI) method for the quantitation of human serum IgG antibodies to Helicobacter pylori. Patients’ sera were injected into the carrier stream of a FI manifold incorporating an H. pylori immuno-adsorbent reactor. The immuno-adsorbent was prepared by covalently linking H. pylori antigens to periodate oxidized Sepharose CL-4B. Any H. pylori antibody present in the serum is bound to the immuno-sorbent. The bound antibody was quantified by injecting into the carrier stream anti-human IgG peroxidase conjugate followed by TMB/H₂O₂ as the enzyme substrate. The FI system was optimized with respect to antigen loading (1.0 mg/ml gel), bioreactor length (3.0 cm), enzyme conjugate (0.2 mg/ml) and sample loop size (250 μl). One hundred clinical samples were analyzed for their H. pylori antibody concentrations and the results evaluated with respect to their receiver-operator characteristics (ROC). When a cut-off absorbance of between 0.7 and 0.75 was used for positive and negative samples a specificity (>95%), a sensitivity (>90%)and an overall accuracy (>94%) were obtained.     

Optimization and validation of a high-performance liquid chromatography method for the analysis of cardiac glycosides in Digitalis lanata

In this study, a simple and reliable HPLC method for the qualitative and quantitative analysis of cardiac glycosides in Digitalis lanata Ehrh. raw material was developed and applied to healthy and phytoplasma-infected plants. The target analytes cover a broad range of secondary metabolites, including primary, secondary and tertiary glycosides and the corresponding aglycones. The sample preparation was carried out by sonication of the plant material with 70% (v/v) aqueous methanol at room temperature, followed by reversed-phase solid-phase extraction purification from interfering pigments. The HPLC analyses were performed on a Symmetry C₁₈ column (75 mm × 4.6 mm I.D., 3.5 μm), with a gradient elution composed of water and acetonitrile, at a flow rate of 1.0 mL/min. The column temperature was set at 20 °C and the photodiode array detector monitored the eluent at 220 nm. The method was validated with respect to ICH guidelines and the validation parameters were found to be highly satisfactory. The application of the method to the analysis of D. lanata leaves indicated that air-drying was the optimum method for raw material processing when compared with freeze-drying. The analysis of healthy and phytoplasma-infected plants demonstrated that the secondary metabolite mainly affected by the pathogen presence was lanatoside C (153.2 μg/100 mg versus 76.1 μg/100 mg). Considering the importance of D. lanata plant material as source of cardiac glycosides, the developed method can be considered suitable for the phytochemical analysis and for the quality assurance of D. lanata used for pharmaceutical purpose.     

Regio- and stereoselective 12-O-demethylation of schizandrin into gomisin T, an important intermediate to gomisin A, by Mortierella sp. (TM-I1104)

A strain TM-I1104 identified as Mortierella sp. was discovered from soil as the most efficient fungus, which converted schizandrin into gomisin T in 91% regioselectivity by microbial 12-O-demethylation. Under optimum conditions, the yield of gomisin T reached around 80%. The faculty of 12-O-demethylation was specific on (+)-schizandrin (natural form) and the optical purity of gomisin T converted from (±)-schizandrin was 96% ee.     

New selective O-debenzylation of phenol with Mg/MeOH

Carboxylate-benzyl and nitro-benzyl groups used in phenols protection were selectively debenzylated with 3-25 equiv of Mg in methanol at room temperature. Good yields of the desired phenols were obtained within 3-10 h from a wide variety of O-(carboxylate-benzyl)- or O-(nitro-benzyl)-phenols. Selective O-debenzylation was possible in the presence of O-(carboxylate-benzyl)- or O-(nitro-benzyl)-phenols with Mg/MeOH.     

Automated determination of folate catabolites in human biofluids (urine,breast milk and serum) by on-line SPE–HILIC–MS/MS

A rapid, precise and fully-automated method for analysis of folate (vitamin B9) and its catabolites (viz. p-aminobenzoylglutamate and its acetamide derivative) in biofluids is here presented. The method is based on on-line hyphenation of solid-phase extraction (SPE) by a Prospekt 2 system with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). The method was analytically characterized by estimation of repeatability (RSD, n = 5, between 0.5 and 4.1%), accuracy (between 96 and 105%), and sensitivity (limits of quantificantion between 0.3 and 8.3 ng/mL (1.1 and 18.8 pmol/mL) or 0.03 and 0.83 ng (0.11 and 1.88 pmol)). The proposed method is suited for routine analysis of folate catabolites as biomarkers to monitor deficiency of vitamin B9.     

Isolation of antioxidants from Psoralea corylifolia fruits using high-speed counter-current chromatography guided by thin layer chromatography-antioxidant autographic assay

A combinative method using high-speed counter-current chromatography (HSCCC) and thin layer chromatography (TLC) as an antioxidant autographic assay was developed to separate antioxidant components from the fruits of Psoralea corylifolia. Under the guidance of TLC bioautography, eight compounds including five flavonoids and three coumarins were successfully separated from the fruits of P. corylifolia by HSCCC with an optimized two-phase solvent system, n-hexane–ethyl acetate–methanol–water (1:1.1:1.3:1, v/v/v/v). The separation produced 5.91 mg psoralen, 6.26 mg isopsoralen, 3.19 mg psoralidin, 0.92 mg corylifol A, and 2.43 mg bavachinin with corresponding purities of 99.5, 99.8, 99.4, 96.4, and 99.0%, as well as three sub-fractions, in a single run from 250 mg ethyl acetate fraction of P. corylifolia extract. Following an additional clean-up step by preparative TLC, 0.4 mg 8-prenyldaidzein (purity 91.7%), 4.18 mg neobavaisoflavone (purity 97.4%) and 4.36 mg isobavachalcone (purity 96.8%) were separated from the three individual sub-fractions. The structures of the isolated compounds were identified by ¹H NMR and ¹³C NMR. The results of antioxidant activity estimation by electron spin resonance (ESR) method showed that psoralidin was the most active antioxidant with an IC₅₀ value of 44.7 μM. This is the first report on simultaneous separation of eight compounds from P. corylifolia by HSCCC.     

High dielectric constant PrYxOy sensing films electrolyte-insulator-semiconductor pH-sensor for the detection of urea

In this paper, we describe the structural and sensing properties of high-k PrY_xO_y sensing films deposited on Si substrates through reactive co-sputtering. Secondary ion mass spectrometry and atomic force microscopy were employed to analyze the compositional and morphological features of these films after annealing at various temperatures. The electrolyte-insulator-semiconductor (EIS) device incorporating a PrY_xO_y sensing membrane that had been annealed at 800 °C exhibited good sensing characteristics, including a high sensitivity (59.07 mV pH⁻¹ in solutions from pH 2 to 12), a low hysteresis voltage (2.4 mV in the pH loop 7 → 4 → 7 → 10 → 7), and a small drift rate (0.62 mV h⁻¹ in the buffer solution at pH 7). The PrY_xO_y EIS device also showed a high selective response towards H⁺. This improvement can be attributed to the small number of crystal defects and the large surface roughness. In addition, the enzymatic EIS-based urea biosensor incorporating a high-k PrY_xO_y sensing film annealed at 800 °C allowed the potentiometric analysis of urea, at concentrations ranging from 1 to 16 mM, with a sensitivity of 9.59 mV mM⁻¹.     

Enzyme mimics of spinel-type CoxNi1−xFe2O4 magnetic nanomaterial for eletroctrocatalytic oxidation of hydrogen peroxide

A series of spinel-type Co_xNi_1−xFe₂O₄ (x = 0, 0.2, 0.4, 0.5, 0.6, 0.8, 1.0) magnetic nanomaterials were solvothermally synthesized as enzyme mimics for the eletroctrocatalytic oxidation of H₂O₂. X-ray diffraction and scanning electron microscope were employed to characterize the composition, structure and morphology of the material. The electrochemical properties of spinel-type Co_xNi_1−xFe₂O₄ with different (Co/Ni) molar ratio toward H₂O₂ oxidation were investigated, and the results demonstrated that Co_0.5Ni_0.5Fe₂O₄ modified carbon paste electrode (Co_0.5Ni_0.5Fe₂O₄/CPE) possessed the best electrocatalytic activity for H₂O₂ oxidation. Under optimum conditions, the calibration curve for H₂O₂ determination on Co_0.5Ni_0.5Fe₂O₄/CPE was linear in a wide range of 1.0 × 10⁻⁸–1.0 × 10⁻³ M with low detection limit of 3.0 × 10⁻⁹ M (S/N = 3). The proposed Co_0.5Ni_0.5Fe₂O₄/CPE was also applied to the determination of H₂O₂ in commercial toothpastes with satisfactory results, indicating that Co_xNi_1−xFe₂O₄ is a promising hydrogen peroxidase mimics for the detection of H₂O₂.     

Determination of flavonoids in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. by capillary electrophoresis with electrochemical detection

Four flavonoids (rutin, hyperoside, quercitrin and quercetin) in Houttuynia cordata Thunb. and Saururus chinensis (Lour.) Bail. were determined by capillary electrophoresis with wall-jet amperometric detection. The working electrode was a 500 μm diameter carbon disc electrode and the detection potential was +0.95 V (versus Ag/AgCl). Effects of several important factors, such as the running buffer and its corresponding pH and concentration, separation voltage, injection time were investigated to acquire the optimum conditions for separation of these four flavonoids. Baseline separation for the four flavonoids was obtained within 21 min in a 60 cm length capillary at a separation voltage of 15 kV with a 60 mmoL/L Na₂B₄O₇-120 mmoL/L NaH₂PO₄ buffer (pH 8.8) as running buffer. The relationship between peak currents and analyte concentrations was linear over about two orders of magnitude with detection limits (defined as S/N = 3) ranging from 0.02 to 0.05 μg/mL for all analytes. This method was applied for the determination of the above four flavonoids in H. cordata Thunb. and S. chinensis (Lour.) Bail. with simple extraction procedures, and the assay results were satisfactory.     

Efficient synthesis of 5′-O(N)-carbamyl and -polycarbamyl nucleosides

A simple and efficient method for the preparation of 5′-O(N)-carbamyl and 5′-O(N)-polycarbamyl nucleoside derivatives is reported. The method consisted of treatment of 2′,3′-O-protected purine (Ado, Ino) or pyrimidine nucleosides (Thd, Urd) with trichloroacetylisocyanate, followed by cleavage of the trichloroacetyl moiety by silica-gel promoted methanolysis during column chromatography. Iterative application of this method gave mono, di, and tricarbamyl derivatives in good to excellent yields (ave = 80%).     

Determination of madecassoside and asiaticoside contents of C. asiatica leaf and C. asiatica-containing ointment and dentifrice by HPLC-coupled pulsed amperometric detection

The present study reports the development and application of an HPLC-coupled pulsed amperometric detection method to determine the madecassoside and asiaticoside contents of Centella asiatica leaf and of commercial C. asiatica-containing ointment and dentifrice. C. asiatica, which was not pretreated, was extracted with 50% ethanol for 10 min. Madecassoside and asiaticoside were separated on a C18 column within 5 min using 25% (v/v) acetonitrile as the mobile phase. Both compounds were detected with high sensitivity when sodium hydroxide was used as a post-column eluent. Madecassoside and asiaticoside both displayed limits of detection of 0.005 μg/mL and linear regression coefficients of 0.9994 and 1.0000, respectively. The intra- and inter-day precisions were < 8.85% and average recovery was > 94.79%. The madecassoside and asiaticoside contents of ointment and dentifrice were successfully determined without sample purification or concentration owing to the high method sensitivity and selectivity.     

Characterization and evaluation of a macroporous adsorbent for possible use in the expanded bed adsorption of flavonoids from Ginkgo biloba L.

The suitability of the use of macroporous adsorbent Amberlite XAD7HP in expanded bed adsorption processes for the isolation of flavonoids from crude extracts of Ginkgo biloba L. has been assessed. The expansion and hydrodynamic properties of expanded beds were investigated and analyzed. The bed expansion as a function of operational fluid velocity was measured and correlated with the Richardson–Zaki equation. Theoretical predictions of the correlation parameters (the terminal settling velocity u_t and exponent n) were improved by modifying equations in the literature. Residence time distributions (RTDs) were studied using acetone as a tracer. Three measures of liquid phase dispersion (the height equivalent of theoretical plate, Bodenstein number and axial distribution coefficient) were investigated and compared to values previously obtained with commercial EBA adsorbents developed for protein purification. A suitable bed expansion ratio was found to be 1.25 times the settled bed height, which occurred at a corresponding flow velocity of 183 cm/h. For an initial settled bed height of 42 cm, the mean residence time of liquid in the expanded bed was around 28 min. Under these flow conditions, the axial mixing coefficient D_ax was 7.54 × 10⁻⁶ m²/s and the Bodenstein number was 28; the number of theoretical plates (N) was 19 and the height equivalent of a theoretical plate (HETP) was 2.77 cm. Rutin trihydrate was used as a model flavonoid for the characterization of the adsorption properties of Amberlite XAD7HP. Adsorption was observed to reach equilibrium within 3 h with 70% of the adsorption capacity being achieved within 30 min. The estimated maximum equilibrium adsorption capacity for rutin was estimated to be 43.0 mg/(g resin) when the results were fitted to Langmuir isotherms. The adsorption performance was not seriously impaired by the physical presence of G. biloba leaf powders. Assessment of the kinetics of the adsorption of rutin revealed that the rate constant for adsorption was only reduced by 15% in the presence of leaf powders at a concentration of 50 mg/mL. The results demonstrated that Amberlite XAD7HP should be suitable for expanded bed adsorption of flavonoids from crude extracts of G. biloba L.     

A colloidal suspension of nanostructured poly(N-butyl benzimidazole)-graphene sheets with high oxidase yield for analytical glucose and choline detections

A colloidal suspension of nanostructured poly(N-butyl benzimidazole)-graphene sheets (PBBIns-Gs) was used to modify a gold electrode to form a three-dimensional PBBIns-Gs/Au electrode that was sensitive to hydrogen peroxide (H₂O₂) in the presence of acetic acid (AcOH). The positively charged nanostructured poly(N-butyl benzimidazole) (PBBIns) separated the graphene sheets (Gs) and kept them suspended in an aqueous solution. Additionally, graphene sheets (Gs) formed “diaphragms” that intercalated Gs, which separated PBBIns to prevent tight packing and enhanced the surface area. The PBBIns-Gs/Au electrode exhibited superior sensitivity toward H₂O₂ relative to the PBBIns-modified Au (PBBIns/Au) electrode. Furthermore, a high yield of glucose oxidase (GOD) on the PBBIns-Gs of 52.3 mg GOD per 1 mg PBBIns-Gs was obtained from the electrostatic attraction between the positively charged PBBIns-Gs and negatively charged GOD. The non-destructive immobilization of GOD on the surface of the PBBIns-Gs (GOD-PBBIns-Gs) retained 91.5% and 39.2% of bioactivity, respectively, relative to free GOD for the colloidal suspension of the GOD-PBBIns-Gs and its modified Au (GOD-PBBIns-Gs/Au) electrode. Based on advantages including a negative working potential, high sensitivity toward H₂O₂, and non-destructive immobilization, the proposed glucose biosensor based on an GOD-PBBIns-Gs/Au electrode exhibited a fast response time (5.6 s), broad detection range (10 μM to 10 mM), high sensitivity (143.5 μA mM⁻¹ cm⁻²) and selectivity, and excellent stability. Finally, a choline biosensor was developed by dipping a PBBIns-Gs/Au electrode into a choline oxidase (ChOx) solution for enzyme loading. The choline biosensor had a linear range of 0.1 μM to 0.83 mM, sensitivity of 494.9 μA mM⁻¹ cm⁻², and detection limit of 0.02 μM. The results of glucose and choline measurement indicate that the PBBIns-Gs/Au electrode provides a useful platform for the development of oxidase-based biosensors.