The fluorescence quenching of Ru(bipy)32+: an application for the determination of bilirubin in biological samples

A simple, sensitive and efficient fluorescence method has been established for the quantitative analysis of bilirubin. The fluorometric determination method was based on the kinetic quenching of ruthenium(II) fluorescence. The quenching effect may be due to the complexation reaction of bilirubin with ruthenium(II). Therefore, the effects of ruthenium concentrations and different surfactants have been studied. Under the optimized experimental parameters, the fluorescence intensity decreased proportionally with the bilirubin concentration and linearity was established in the range of 3.3 × 10⁻⁷ to 3.0 × 10⁻⁴ M bilirubin. The detection limit calculated from the calibration graph was found to be 5.2 × 10⁻⁸ M. The relative standard deviation (RSD) of 10 consecutive measurements of 8.0 × 10⁻⁶ M bilirubin was 3.0%, while the recoveries of bilirubin in both human serum and urine samples were obtained in the range 94.0–99.5%. The interference study shows that the developed fluorescence based technique is fast, easy to carry out and shows negligible interference. The developed technique was successfully applied for the analysis of bilirubin in human urine and serum samples. All the experimental results and quality parameters confirmed the sensitivity and reproducibility of the proposed technique for bilirubin determination in human urine and serum samples .     

Synthesis and characterization of phenothiazine sensor for spectrophotometric and fluorescence detection of cyanide

A sensitive and selective phenothiazine-based sensor (PTZ) has been successfully synthesized. The sensor PTZ displayed specific identification of CN⁻ ‘turn-off’ fluorescence responses with a quick reaction and strong reversibility in an acetonitrile:water (90:10, V/V) solution. The sensor PTZ for detecting CN⁻ exhibits the marked advantages of quenching the fluorescence intensity, fast response time (60 s), and low value of the detection limit. The concentration that is authorized for drinking water by the WHO (1.9 μM) is far higher than the detection limit, which was found to be 9.11 × 10⁻⁹. The sensor displays distinct colorimetric and spectrofluorometric detection for CN⁻ anion due to the addition of CN⁻ anion to the electron-deficient vinyl group of PTZ, which reduces intramolecular charge transfer efficiencies. The 1:2 binding mechanism of PTZ with CN⁻ was validated by fluorescence titration, Job's plot, HRMS, ¹H NMR, FTIR analysis, and density functional theory (DFT) investigations, among other methods. Additionally, the PTZ sensor was successfully used to precisely and accurately detect cyanide anions in actual water samples.     

A ratiometric fluorescence strategy based on copper nanoclusters/carbon dots for sensitive detection of doxorubicin

Sensitive detection of doxorubicin (DOX) is critical for clinical theranostics. A novel ratiometric fluorescence strategy based on the inner filter effect (IFE) has been established for the sensitive detection of DOX by designing a ratiometric fluorescence probe. In the presence of DOX, the fluorescence intensity of copper nanoclusters (CuNCs) at 485 nm decreases, and the fluorescence intensity of carbon dots at 560 nm increases. Therefore, DOX can be quantitatively detected by measuring the ratio of the fluorescence intensities at 560 and 485 nm (F₅₆₀/F₄₈₅). The F₅₆₀/F₄₈₅ ratio exhibits a linear correlation with the DOX concentration in the range from 1.0 × 10⁻⁸ M to 1.0 × 10⁻⁴ M with the detection limit of 3.7 nM. Furthermore, this method was also successfully applied to the analysis of DOX in human plasma samples, affording an effective platform for drug safety management.     

Utility of the food colorant erythrosine B as an effective green probe for quantitation of the anticancer sunitinib. Application to pharmaceutical formulations and human plasma

Sunitinib is a tyrosine kinase inhibitor used for the treatment of renal cell carcinoma and gastrointestinal stromal tumors. In this study, two spectroscopic methods, spectrofluorometric and spectrophotometric, were utilized to quantify sunitinib in different matrices. In method I, the native fluorescence of erythrosine B was quenched by forming ion-pair complex with increasing quantities of sunitinib. This approach was utilized for measuring sunitinib in its dosage forms and spiked plasma. After excitation at 528 nm, the quenching of fluorescence is linearly related to the concentration across the range of 0.05–0.5 μg mL⁻¹ at 550 nm in Britton–Robinson buffer (pH 4.0), with a correlation value of 0.9999 and a high level of sensitivity with detection limit down to 10 ng mL⁻¹. Method II relies on spectrophotometric measurements of the produced complex at 550 nm across a range of 0.5–10.0 μg mL⁻¹, with good correlation value of 0.9999. This method has a detection limit down to 0.16 μg mL⁻¹. The proposed methodologies were validated according to International Conference on Harmonization (ICH) guidelines with satisfactory results. The stoichiometry of the reaction was determined through the application of Job's method, while the mechanism of quenching was investigated by employing the Stern–Volmer plot. The designated methods were used to estimate sunitinib in its capsules and in spiked human plasma. Additionally, the statistical analysis of the data revealed no substantial differences when compared to previous reported spectroscopic method. Green assessment tools provide further details about the eco-friendly nature of the methods.     

Investigating Fluorescence Quenching of ZnS Quantum Dots by Silver Nanoparticles

Water dispersible zinc sulfide quantum dots (ZnS QDs) with an average diameter of 2.9 nm were synthesized in an environment friendly method using chitosan as stabilizing agent. These nanocrystals displayed characteristic absorption and emission spectra having an absorbance edge at 300 nm and emission maxima (λ _emission) at 427 nm. Citrate-capped silver nanoparticles (Ag NPs) of ca. 37-nm diameter were prepared by modified Turkevich process. The fluorescence of ZnS QDs was significantly quenched in presence of Ag NPs in a concentration-dependent manner with K _sv value of 9 × 10⁹ M⁻¹. The quenching mechanism was analyzed using Stern–Volmer plot which indicated mixed nature of quenching. Static mechanism was evident from the formation of electrostatic complex between positively charged ZnS QDs and negatively charged Ag NPs as confirmed by absorbance study. Due to excellent overlap between ZnS QDs emission and surface plasmon resonance band of Ag NPs, the role of energy transfer process as an additional quenching mechanism was investigated by time-resolved fluorescence measurements. Time-correlated single-photon counting study demonstrated decrease in average lifetime of ZnS QDs fluorescence in presence of Ag NPs. The corresponding F?rster distance for the present QD–NP pair was calculated to be 18.4 nm.     

Newly synthesized Fe-doped CDs for colorimetric and fluorometric nanozyme-based levodopa sensing

A simple single-pot hydrothermal method was used to fabricate a Fe, N, and S co-doped carbon dots (Fe-CDs) nanozyme using ferric chloride and sunset yellow as precursors. The fabricated Fe-CDs exhibited intense green fluorescence at 460 nm with excitation-independent properties and a high quantum yield of 40.23%. This nanozyme mimics peroxidase by catalyzing the oxidation of tetramethylbenzidine (TMB) by H₂O₂ to yield a blue-coloured TMB_ox product at 652 nm. Dual detection methods were established for determining levodopa (l -dopa) by taking advantage of the high nanozyme activity and the distinct fluorescence aspect. Both determination methods are based on the oxidation of l -dopa by H₂O₂ in the presence of Fe-CDs and fading of the blue colour of the TMB_ox. The colorimetric method monitors the amount of colour fading of TMB_ox. In the fluorometric method, the formed blue TMB_ox absorbs the emission light of the Fe-CDs; when l -dopa is present, this effect decreases and the intensity of the emission light increases. The nanozyme-based detection procedures exhibit good linearity in the ranges 2.17 × 10⁻³ to 34.78 × 10⁻³ mM [limit of detection (LOD) = 0.84 × 10⁻³ mM] and 0.85 × 10³ to 16.95 × 10³ nM (LOD = 0.102 × 10³ nM) for colorimetric and fluorometric methods, respectively.     

Utilization of erythrosine B in spectrofluorimetric quenching analysis of amlodipine and perindopril in pharmaceutical and biological matrices

A spectrofluorimetric approach that is sensitive, simple, validated, and cost-effective has been proposed for the estimation of amlodipine (AML) and perindopril (PER) in their bulk powders, pharmaceutical formulations, and spiked human plasma. The recommended approach utilized the quantitative quenching effect of the two cited drugs on the fluorescence intensity of erythrosine B, as a result of complex binary reactions among each drug with erythrosine B at pH 3.5 (Teorell and Stenhagen buffer). The quenching of erythrosine B fluorescence was recorded at 554 nm after excitation at 527 nm. The calibration curve was detected in the range 0.25–3.0 μg ml⁻¹, with a correlation coefficient of 0.9996 for AML, and 0.1–1.5 μg ml⁻¹, with a correlation coefficient of 0.9996 for PER. The established spectrofluorimetric approach was validated for the estimation of the cited drugs with high sensitivity regarding International Council on Harmonization guidelines. Therefore, the established approach could be utilized for quality control of the cited drugs in their pharmaceutical formulations.     

A novel spectrofluorimetric method for determination of lomefloxacin adopting on zinc(II) chelation strategy: Application in human plasma

A new sensitive and instantaneous spectrofluorimetric method for efficient determination of lomefloxacin (LMX) in its pure, dosage form and human plasma was designed. The developed method depends on formation of a metal-chelation compound of LMX as a ligand with zinc(II) in a buffer of acetate (pH 5.5). The following parameters; type of metal, concentration of metal, pH, type of buffer and diluting solvent were optimized. After carefully investigation; 0.2 mM zinc, 2.0 ml acetate buffer (pH 5.5) and water as diluting solvent were set as optimum reaction conditions. Under these conditions, a large increase in the intensity of the fluorescence of LMX was attained at 450 after excitation at 284 nm. The limits of detection and quantification were 5.8 and 1.9 ng ml⁻¹, respectively, with linearity range of 10.0 to 500.0 ng ml⁻¹. The binding mode of LMX and zinc(II) ion (Zn²⁺) was found to be 2:1, respectively, and confirmed by Job's plot method. Furthermore, it extended to the analysis of LMX in the spiked plasma of humans with percentage recovery (98.70 ± 0.97 to 100.30 ± 1.69%, n = 3).     

A ‘turn-off’ fluorescence sensor for selective Hg(II) based on phenothiazine derivative

Given how crucial it is to preserve a human-safe and sustainable environment, the rapid discovery of possibly lethal heavy metals such as Hg(II) has drawn much attention in recent years. A novel sensor, known as (E)-2-((10-octyl-10H-phenothiazin-3-yl)methylene)hydrazine-1-carbothioamide (PTZHC), was synthesized as a fluorescence ‘on–off’ sensor for Hg²⁺ ions. Coordination alters organic molecule electron densities, quenching the fluorescence intensity. PTZHC was described completely with the help of FTIR and ¹H-NMR spectrum studies. The Hg²⁺ ion was successfully detected using the PTZHC sensor even when there were other metal ions present. The limit of the detection was estimated to be 2.5 × 10⁻⁸ M and the Job's plot examination implied that PTZHC was bound to Hg²⁺ with a simple 1:1 stoichiometry in s CH₃CN/H₂O (9:1, v/v) suspension. To further cast light on the bridged effect on geometric and optoelectronic characteristics, time-dependent density functional theory (TD-DFT) at the B3LYP/6-31G(d) level and DFT were both examined.     

Study of the interaction of human serum albumin with Alstonia scholaris leaf extract-mediated silver nanoparticles having bactericidal property

This study investigates the green synthesis of AgNPs from 1 mM aqueous AgNO₃ using 10% leaf extract of Alstonia scholaris (Chhatim) for its wide antibacterial and medicinal properties. The synthesized AgNPs were duly characterized by UV–vis (UV–vis) spectrophotometry, dynamic light scattering, field emission scanning electron microscopy, transmission electron microscopy, energy-dispersive analysis of X-rays spectroscopy, and fourier transform infrared spectrophotometry. Their antibacterial property was tested against Escherichia coli (ATCC 25922), and minimum inhibitory concentrations of 0.08 nM of AgNPs were obtained, which suggests improved therapeutic efficacy. We report the interaction of human serum albumin (HSA) with this nanoparticle, and this interaction was studied by UV–vis, fluorescence, and circular dichroism spectroscopies and zeta potential measurement at room temperature. It was found that the AgNPs form a complex with HSA, which may cause the slightest change in the conformation of HSA. The calculated values of Stern-Volmer quenching constant, binding constant, and binding distance were 1.82 × 10⁷ M⁻¹, 1.58 × 10⁷ M⁻¹, and 3.68 nm, respectively. Therefore, in future, the present study may provide useful information to design a better antibacterial compound by using green synthesized nanoparticles with fewer side effects.     

Near-infrared triple-helical peptide with quenched fluorophores for optical imaging of MMP-2 and MMP-9 proteolytic activity in vivo

The gelatinase members of the MMP family have consistently been associated with tumor invasiveness, which make them an attractive target for molecular imaging. We report new activatable proteolytic optical imaging agents that consist of triple-helical peptide (THP) conjugates, with high specificity to the gelatinases, bearing quenched cypate dyes. With quenching efficiencies up to 51%, the amplified fluorescence signal upon cypate₃-THP hydrolysis by the gelatinases (k_cat/K_M values of 6.4 × 10³ M⁻¹ s⁻¹ to 9.1 × 10³ M⁻¹ s⁻¹ for MMP-2 and MMP-9, respectively) in mice bearing human fibrosarcoma xenografted tumors was monitored with fluorescence molecular tomography. There was significant fluorescence enhancement within the tumor and this enhancement was reduced by treatment with pan-MMP inhibitor, Ilomastat. These data, combined with the gelatinase substrate specificity observed in vitro, indicated the observed fluorescence at the site of the tumor was due to gelatinase mediated hydrolysis of cypate₃-THP.     

Cationic effect of imidazolium-based ionic liquid on the stability of myoglobin

The conformational change of myoglobin (Mb) during guanidine hydrochloride (GuHCl)-induced protein unfolding in the presence of various ionic liquids (ILs) in phosphate buffer was investigated using both the Soret band absorption and the fluorescence of tryptophan measurements. The GuHCl-induced denaturation midpoints of Mb derived from the absorption and fluorescence spectra were almost similar in the presence of 150 mM ILs with the same cation 1-butyl-3-methylimidazolium (Bmim⁺) but different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer. In addition, the denaturation midpoints of Mb in the presence of ILs were little lower than those in the absence of ILs in phosphate buffer. For the sake of clarity and comparison, we also measured the GuHCl-induced denaturation midpoints of Mb in the presence of 150 mM sodium salts with different anions (BF₄⁻, NO₃⁻, Cl⁻, and Br⁻) in phosphate buffer and found that their corresponding denaturation midpoints of Mb were almost similar to those observed in the absence of sodium salts in phosphate buffer. These experimental data indicate that Bmim⁺ cation can promote the unfolding of Mb. Further experiments revealed that the denaturation ability of ILs increases with increasing alkyl chain length of imidazolium cation of ILs and that hydroxyl-substituted imidazolium cation could also promote the unfolding of Mb.     

Sensitive spectrofluorometric method for the determination of ascorbic acid in pharmaceutical nutritional supplements using acriflavine as a fluorescence reagent

An easily performed, specific, sensitive, rapid, reliable and inexpensive procedure for the spectrofluorometric quantitation of ascorbic acid was proposed using acriflavine as a fluorescence quenching reagent. The procedure was based on the determined quenching effect of ascorbic acid on the natural fluorescence signal of acriflavine and the reaction between ascorbic acid and acriflavine in Britton–Robinson buffer solution (pH 6) to produce an ion‐associated complex. The reduction in acriflavine fluorescence intensity was detected at 505 nm, while excitation occurred at 265 nm. The relationship between quenching fluorescence intensity (?F) and concentration of ascorbic acid was linear (R² = 0.9967) within the range 2–10 μg/ml and with a detection limit of 0.08 μg/ml. No significant interference was detected from other materials often found in pharmaceutical nutritional tablets. The obtained results were compared with those from high‐performance liquid chromatography and appeared in good agreement, with no important differences in precision or accuracy. The proposed spectrofluorimetric method was used to determine the amount of ascorbic acid in a number of commercial pharmaceutical nutritional supplement tablets with a 95% confidence performance.     

Highly sensitive spectrofluorimetric determination of trace amounts of prulifloxacin using the aluminium(III)–prulifloxacin system

The fluorescence of the prulifloxacin (PUFX)–Al(III) system was investigated . Experiments indicated that the fluorescence intensity of prulifloxacin could be greatly enhanced by Al(III) and sensitized by sodium dodecylbenzene sulphonate (SDBS). Accordingly, a sensitive spectrofluorimetric method for the determination of prulifloxacin was established. While excited at 275 nm, the enhanced fluorescence intensity at 412 nm of the system (ΔF) showed a good linear relationship with the concentration of prulifloxacin within the range 4.0 × 10^–8–3.0 × 10^–6 mol/L. The regression equation was ΔF = 9.83 + 10.8 × 10⁷c (mol/L); the correlation coefficient and detection limit (3σ/k) were 0.99901 and 2.0 × 10^–8 mol/L, respectively. The proposed method has been successfully applied to determine prulifloxacin in real pharmaceutical samples. The luminescence mechanism of the system is also discussed in detail. Copyright © 2008 John Wiley & Sons, Ltd.     

PROPERTIES OF TYROSINE HYDROXYLASE IN PERIPHERAL NERVES

Approximately 80 per cent of tyrosine hydroxylase activity in bovine mandibular nerve and rabbit sciatic nerve was soluble, and the rest of the activity was particle-bound. The soluble enzyme in bovine mandibular nerve was isolated by ammonium sulphate fractionation (25–35 per cent saturation). The enzyme had a pH optimum at 5·9 in Tris-acetate buffer, and at 6·5 in Tris-HCl or phosphate buffer. The enzyme required a tetrahydropteridine cofactor. K_m values toward various tetrahydropteridines such as l -erythro-tetrahydrobiopterin (a probable natural cofactor), 2-amino-4-hydroxy-6-methyltetrahydropteridine, and 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine were 2 × 10⁻⁵m , 5 × 10⁻⁵m and 4 × 10⁻⁴m , respectively. The K_m value for tyrosine at 1 × 10⁻³m -2-amino-4-hydroxy-6-methyltetrahydropteridine as a cofactor was 5 × 10⁻⁵m . The enzyme activity was markedly stimulated with Fe²⁺ or catalase, but Fe²⁺ gave higher activity. The activity was inhibited with α, α′-dipyridyl, l -α-methyl-p-tyrosine, and various catecholamines. Among catecholamines, dopamine was the most potent inhibitor. l -5-Hydroxytryptophan was an inhibitor as potent as dopamine. Neither d -5-hydroxytryptophan nor 5-hydroxytryptamine inhibited the enzyme. The inhibition by l -5-hydroxytryptophan was partially competitive with tetrahydrobiopterin at concentrations higher than 9 × 10⁻⁵m , and partially uncompetitive at concentrations lower than 9 × 10⁻⁵m . The addition of heparin or lysolecithin did not affect enzyme activity with tetrahydrobiopterin as cofactor.     

A new benzimidazole‐based selective and sensitive ‘on–off’ fluorescence chemosensor for Cu2+ ions and application in cellular bioimaging

Two new twinborn benzimidazole derivates ( L and A ), which bonded pyridine via the ester space on the opposite and adjacent positions of the benzene ring of benzimidazole respectively, were designed and synthesized. Compound L displayed fluorescence quenching response only towards copper(II) ions (Cu²⁺) in acetonitrile solution with high selectivity and sensitivity. However, compound A presented ‘on–off’ fluorescence response towards a wide range of metal ions to different degrees and did not have selectivity. Furthermore, compound L formed a 1:1 complex with Cu²⁺ and the binding constant between sensor L and Cu²⁺ was high at 6.02 × 10⁴ M^?1. Job's plot, mass spectra, IR spectra, ¹H‐NMR titration and density functional theory (DFT) calculations demonstrated the formation of a 1:1 complex between L and Cu²⁺. Chemosensor L displayed a low limit of detection (3.05 × 10^?6 M) and fast response time (15 s) to Cu²⁺. The Stern–Volmer analysis illustrated that the fluorescence quenching agreed with the static quenching mode. In addition, the obvious difference of L within HepG2 cells in the presence and absence of Cu²⁺ indicated L had the recognition capability for Cu²⁺ in living cells.     

Determination of pioglitazone hydrochloride by flow injection chemiluminescence tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex system

A novel flow injection-chemiluminescence (FI–CL) approach is proposed for the assay of pioglitazone hydrochloride (PG-HCl) based on its enhancing influence on the tris(2,2′-bipyridyl)ruthenium(II)–silver(III) complex (Ru(bipy)₃²⁺-DPA) CL system in sulfuric acid medium. The possible CL reaction mechanism is discussed with CL and ultraviolet (UV) spectra. The optimum experimental conditions were found as: Ru(bipy)₃²⁺, 5.0 × 10⁻⁵ M; sulfuric acid, 1.0 × 10⁻³ M; diperiodatoargentate(III) (DPA), 1.0 × 10⁻⁴ M; potassium hydroxide, 1.0 × 10⁻³ M; flow rate 4.0 ml min⁻¹ for each flow stream and sample loop volume, 180 μl. The CL intensity of PG-HCl was linear in the range of 1.0 × 10⁻³ to 5.0 mg L⁻¹ (R² = 0.9998, n = 10) with limit of detection [LOD, signal-to-noise ratio (S/N) = 3] of 2.2 × 10⁻⁴ mg L⁻¹, limit of quantification (LOQ, S/N = 10) of 6.7 × 10⁻⁴ mg L⁻¹, relative standard deviation (RSD) of 1.0 to 3.3% and sampling rate of 106 h⁻¹. The methodology was satisfactorily used to quantify PG-HCl in pharmaceutical tablets with recoveries ranging from 93.17 to 102.77 and RSD from 1.9 to 2.8%.     

Nuclear fast red‐based colorimetric sensors for sensitive and selective detection of Ag ions

The reduction of nuclear fast red (NFR) stain by sodium tetrahydroboron was catalyzed in the presence of silver ions (Ag⁺). The fluorescence properties of reduced NFR differed from that of NFR. The product showed fluorescence emission at 480 nm with excitation at 369 nm. Furthermore, the fluorescence intensity of the mixture increased strongly in the presence of Ag⁺ and Britton–Robinson buffer at pH 4.78. There was a good linear relationship between increased fluorescence intensity (ΔI) and Ag⁺ concentration in the range 5.0 × 10^?9 to 5.0 × 10^?8 M. The correlation coefficient was 0.998, and the detection limit (3σ/k) was 1.5 × 10^?9 M. The colour of the reaction system changed with variation in Ag⁺ concentration over a wide range. Based on the colour change, a visual semiquantitative detection method for recognition and sensing of Ag⁺ was developed for the range 1.0 × 10^?8 to 5.0 × 10^?4 M, with an indicator that was visible to the naked eye. Therefore, a sensitive, simple method for determination of Ag⁺ was developed. Optimum conditions for Ag⁺ detection, the effect of other ions and the analytical application of Ag⁺ detection of synthesized sample were investigated.     

Amperometric xanthine biosensors based on electrodeposition of platinum on polyvinylferrocenium coated Pt electrode

Novel xanthine biosensors were successfully fabricated by immobilizing xanthine oxidase on polyvinylferrocenium perchlorate matrix (PVF⁺ClO₄⁻) and platinum electrodeposited polyvinylferrocenium perchlorate matrix. PVF⁺ClO₄⁻ film was coated on Pt electrode at +0.7 V vs. Ag/AgCl by electrooxidation of polyvinylferrocene (PVF). Platinum nanoparticles were deposited on PVF⁺ClO₄⁻ electrode by electrochemical deposition in 2.0 mM H₂PtCl₆ solution at −0.2 V. Xanthine oxidase was incorporated into the polymer matrix via ion exchange process by immersing modified Pt electrodes in the enzyme solution. The amperometric responses of the biosensors were measured via monitoring oxidation current of hydrogen peroxide at +0.5 V. Under the optimal conditions, the linear ranges of xanthine detection were determined as 1.73 × 10⁻³–1.74 mM for PVF⁺XO⁻ and 0.43 × 10⁻³–2.84 mM for PVF⁺XO⁻/Pt. The detection limits of xanthine were 5.20 × 10⁻⁴ mM for PVF⁺XO⁻ and 1.30 × 10⁻⁴ mM for PVF⁺XO⁻/Pt. Moreover, the effects of applied potential, electrodeposition potential, H₂PtCl₆ concentration, amount of electrodeposited Pt nanoparticles, thickness of polymeric film, temperature, immobilization time, xanthine and xanthine oxidase concentrations on the response currents of the biosensors were investigated in detail. The effects of interferents, the operational and storage stabilities of biosensors and the applicabilities to drug samples of the biosensors analysis were also evaluated.