Application of Molecularly Imprinted Polymer-modified Potentiometric Sensor for Quantitative Determination of Histamine in Serum.

A molecularly imprinted polymer-modified potentiometric histamine (HIS) sensor was prepared and used for quantitative determination of HIS in bovine serum. The calibration curve using the potential responses measured in 1 × 10-3 mol L-1 phosphate buffer (pH 7.4) showed good linearity in the HIS concentration range of 3 × 10-4 to 1 × 10-2 mol L-1 (r = 0.92), with a detection limit of 1.6 × 10-4 mol L-1. In bovine serum samples, the HIS sensor showed good recovery values of 91 - 104%. Therefore, this HIS sensor successfully determined the HIS concentration in bovine serum samples.

Antihypertensive constituents in Sanoshashinto.

It has been reported that Sanoshashinto (SanHuangXieXinTang, ), which is composed of Rhei Rhizoma, Scutellariae Radix, and Coptidis Rhizoma, exhibits vasorelaxant effects in vitro and lowers blood pressure of patients. Based on this discovery, in this study, a mixture containing those three materials and combinations of them were extracted with methanol, and the extracts were fractionated into different parts. Effects of all extracts and fractions on high concentration of potassium chloride (High K+)- or noradrenaline (NA)-induced contractions of isolated rat aortic rings or helical strips were examined. Qualitative and quantitative HPLC analyses of the extracts and the fractions revealed that the contents of baicalin and berberine in Sanoshashinto methanol extract (SHXXTM) were higher than those of the other constituents. All pharmacological and HPLC data were analyzed by principal component analysis (PCA) software and the results indicated that baicalin, berberine, palmatine, baicalein, and wogonoside contributed significantly to the pharmacological activity. Furthermore, spontaneously hypertensive rats (SHRs) that were orally given SHXXTM or a baicalin-berberine combination showed significantly reduced increase in the rate of systolic blood pressure (SBP) compared to the control group. These findings suggested that Sanoshashinto has significant vasorelaxant effects in vitro and antihypertensive effects in vivo, and baicalin and berberine, which were the principal constituents of Scutellariae Radix and Coptidis Rhizoma, were the main antihypertensive constituents in Sanoshashinto. It was speculated that baicalin and berberine produced vasorelaxant effects by activating the NO/cGMP pathway and that the BKCa channel and the DAG/PKC/CPI-17 pathway were also involved.

Effects of mono- and dialkylglucosides on the characterisation and blood circulation of lipid nanoemulsions.

Aim: Effects of two cosurfactants, n-alkylglycosides with mono- or disaccharide groups - N-nonyl ß-D-glucopyranoside (N-Glu) and N-decyl ß-D-maltoside (D-Mal) - were studied to the stability in saline solution, interaction with serum albumin, and blood circulation of the lipid nanoemulsion (LNE).Methods: The LNEs composed of soybean oil, phosphatidylcholine, and sodium palmitate were prepared without (Control-LNE) and with N-Glu or D-Mal (NG-LNE and DM-LNE, respectively).Results: In saline solution, NG-LNE exhibited a smaller droplet size than Control-LNE, while the size of DM-LNE was significantly increased compared with the other LNEs. The fluorescence resonance energy transfer method showed that the order of albumin interaction was DM-LNE > NG-LNE > Control-LNE. In vivo blood circulation in mice, showed greater fractions of both NG-LNE and DM-LNE remaining in blood over time compared with Control-LNE.Conclusions: The nature of high stability in saline solution and high affinity for serum albumin led to the prolonged circulation of LNE.

A Molecularly Imprinted Polymer-modified Potentiometric Sensor for the Detection of Glutathione.

Potentiometric glutathione (GSH) sensors were fabricated using a molecularly imprinted polymer prepared from GSH, methacrylic acid (MAA), and ethylene glycol dimethacrylate as the template molecule, functional monomer, and cross-linker, respectively. Five GSH sensors were prepared with different ratios of GSH to MAA. Their potential responses were measured in a GSH aqueous solution using Ag/AgCl as the reference electrode. A GSH sensor prepared with a GSH:MAA ratio of 2:32 had the best responsivity, while the sensor synthesized from a non-imprinted polymer prepared without GSH (NIP sensor) showed a potential response value of almost zero after the addition of GSH. The ratio of the potential responses of the GSH sensor to the NIP sensor was 8.21. Additionally, the GSH sensor had good linearity over a GSH concentration range of 1 × 10-5 to 2 × 10-4 mol L-1. The GSH sensor provided good quantification and high specificity for GSH and is expected to be applicable for easy and direct determinations of GSH.

Combination of 1H nuclear magnetic resonance spectroscopy and principal component analysis to evaluate the lipid fluidity of flutamide-encapsulated lipid nanoemulsions.

The lipid fluidity of various lipid nanoemulsions (LNEs) without and with flutamide (FT) and containing one of two neutral lipids, one of four phosphatidylcholines as a surfactant, and sodium palmitate as a cosurfactant was investigated by the combination of 1H nuclear magnetic resonance (NMR) spectroscopy and principal component analysis (PCA). In the 1H NMR spectra, the peaks from the methylene groups of the neutral lipids and surfactants for all LNE preparations showed downfield shifts with increasing temperature from 20 to 60 °C. PCA was applied to the 1H NMR spectral data obtained for the LNEs. The PCA resulted in a model in which the first two principal components (PCs) extracted 88% of the total spectral variation; the first PC (PC-1) axis and second PC (PC-2) axis accounted for 73 and 15%, respectively, of the total spectral variation. The Score-1 values for PC-1 plotted against temperature revealed the existence of two clusters, which were defined by the neutral lipid of the LNE preparations. Meanwhile, the Score-2 values decreased with rising temperature and reflected the increase in lipid fluidity of each LNE preparation, consistent with fluorescence anisotropy measurements. In addition, the changes of Score-2 values with temperature for LNE preparations with FT were smaller than those for LNE preparations without FT. This indicates that FT encapsulated in LNE particles markedly suppressed the increase in lipid fluidity of LNE particles with rising temperature. Thus, PCA of 1H NMR spectra will become a powerful tool to analyze the lipid fluidity of lipid nanoparticles. Graphical abstract ᅟ.

Thermal Behavior of 19F Nuclear Magnetic Resonance Signal of 19F-Containing Compound in Lipid Nano-Emulsion for Potential Tumor Diagnosis.

We developed carriers of a 19F magnetic resonance imaging (19F MRI) agent, capable of responding to the temperature difference for cancer diagnosis. The carriers were based on high melting point (mp) neutral lipids, namely, tripalmitin (TPT) and tristearin (TSR) and triarachidin (TAC). Lipid nano-emulsions (LNEs) containing a fluorine compound, i.e., a modified α-tocopherol (19F-TP), were respectively prepared as TPT-LNE, TSR-LNE, TAC-LNE1, and TAC-LNE2 and studied by 19F NMR spectroscopy. In LNE prepared with soybean oil as a control, the full width at half maximum (FWHM) values of the 19F NMR signal of 19F-TP remained constant at 25, 37, and 42°C, while those of the LNEs prepared from a neutral lipid with a high mp showed a sharp decrease between 25 and 37°C. The magnitude of the decrease followed the order: TPT-LNE < TSR-LNE < TAC-LNE1. However, TAC-LNE2, for which the amount of encapsulated 19F-TP was one third less than that of TAC-LNE1, showed a sharp decline in the FWHM between 37 and 42°C. To examine these changes, the 19F spin-lattice (T1) and spin-spin (T2) relaxation times of 19F-TP were measured. TAC-LNE2 in particular showed a substantial change in its T2 value between 37 and 42°C compared with the change of its T1 value. This result was attributed to activation of the molecular motion of 19F-TP in TAC-LNE2 from 37 to 42°C. Thus, TAC-LNE showed potential for use as a carrier for cancer diagnosis using 19F MRI.

Alpha7 nicotinic acetylcholine receptor-specific agonist DMXBA (GTS-21) attenuates Aß accumulation through suppression of neuronal γ-secretase activity and promotion of microglial amyloid-ß phagocytosis and ameliorates cognitive impairment in a mouse model of Alzheimer's disease.

We previously demonstrated that stimulation of nicotinic acetylcholine receptors (nAChRs) increases amyloid-ß (Aß) phagocytosis in rat microglia and is closely associated with the decrease of brain Aß and amelioration of memory dysfunction in a transgenic mouse model of Alzheimer's disease (AD). Here, we examined the subtypes of nAChRs involved in these beneficial effects. In primary cultures of rat microglia, the α7 nAChR selective agonist 3-[(2,4-dimethoxy)benzylidene]-anabaseine dihydrochloride (DMXBA) promoted Aß and fluorescent latex bead phagocytosis, whereas selective α7 nAChR antagonists suppressed the enhanced Aß phagocytosis. In a transgenic mouse model of AD, administration of DMXBA attenuated brain Aß burden and memory dysfunction. Moreover, DMXBA suppressed γ-secretase activity in solubilized fractions of human neuroblastoma cells and transgenic mouse brain. These results suggested that selective activation of α7 nAChRs promoted microglial Aß phagocytosis and suppressed neuronal γ-secretase activity to contribute to the attenuation of the brain Aß burden and cognitive impairment. Thus, we propose neuronal and microglial α7 nAChRs as new therapeutic targets in the treatment of AD.

Polydiacetylene Liposomal Aequorin Bioluminescent Device for Detection of Hydrophobic Compounds.

In this study, a polydiacetylene liposomal aequorin bioluminescent device (PLABD) that functioned through control of the membrane transport of Ca(2+) ions was developed for detecting hydrophobic compounds. In the PLABD, aequorin was encapsulated in an internal water phase and a calcium ionophore (CI) was contained in a hydrophobic region. Membrane transport of Ca(2+) ions across the CI was suppressed by polymerization between diacetylene molecules. On addition of an analyte, the membrane transport of Ca(2+) ions across the CI increased, and Ca(2+) ions from the external water phase could diffuse into the internal water phase via the CI, which resulted in bioluminescence of the aequorin. Lidocaine, procaine, and procainamide were used as model compounds to test the validity of the detection mechanism of the PLABD. When each analyte was added to a suspension of the PLABD, bioluminescence from the aequorin in the PLABD was observed, and the level of this bioluminescence increased with increasing analyte concentration. There was a linear relationship between the logarithm of the analyte concentration and the bioluminescence for all analytes as follows: R = 0.89 from 10 nmol L(-1) to 10 mmol L(-1) for lidocaine, R = 0.66 from 10 nmol L(-1) to 100 µmol L(-1) for procaine, and R = 0.74 from 100 nmol L(-1) to 100 µmol L(-1) for procainamide. Compared to the traditional colorimetric method using polydiacetylene liposome, the PLABD was superior for both the sensitivity and dynamic range. Thus, PLABD is a valid, simple, and sensitive signal generator for detection of hydrophobic compounds that interact with PLABD membranes.

19F Nuclear Magnetic Resonance Spectrometric Determination of the Partition Coefficients of Flutamide and Nilutamide (Antiprostate Cancer Drugs) in a Lipid Nano-Emulsion and Prediction of Its Encapsulation Efficiency for the Drugs.

To design a useful lipid drug carrier having a high encapsulation efficiency (EE%) for the antiprostate cancer drugs flutamide (FT) and nilutamide (NT), a lipid nano-emulsion (LNE) was prepared with soybean oil (SO), phosphatidylcholine (PC), and sodium palmitate, and the partition coefficients (K ps) of the drugs for the LNE were determined by 19F nuclear magnetic resonance (NMR) spectrometry. The 19F NMR signal of the trifluoromethyl group of both drugs showed a downfield shift from an internal standard (trifluoroethanol) and broadening according to the increase in the lipid concentration due to their interaction with LNE particles. The difference in the chemical shift (Δδ) of each drug caused by the addition of LNE was measured under different amounts of LNE, and the K p values were calculated from the Δδ values. The results showed that FT has higher lipophilicity than NT. The total lipid concentration (SO + PC) required to encapsulate each drug into LNE with an EE% of more than 95% was calculated from the K p values as 93.3 and 189.9 mmol/L for FT and NT, respectively. For an LNE prepared with the total lipid concentration of 215 mmol/L, the predicted EE% values were 98 and 96% for FT and NT, respectively, while the experimental EE% values determined by a centrifugation method were approximately 99% for both drugs. Thus, the 19F NMR spectrometric method is a useful technique to obtain the K p values of fluorinated drugs and thereby predict the theoretical lipid concentrations and prepare LNEs with high EE% values.

Partitioning of organophosphorus pesticides into phosphatidylcholine small unilamellar vesicles studied by second-derivative spectrophotometry.

In order to quantitatively examine the lipophilicity of the widely used organophosphorus pesticides (OPs) chlorfenvinphos (CFVP), chlorpyrifos-methyl (CPFM), diazinon (DZN), fenitrothion (FNT), fenthion (FT), isofenphos (IFP), profenofos (PFF) and pyraclofos (PCF), their partition coefficient (Kp) values between phosphatidylcholine (PC) small unilamellar vesicles (SUVs) and water (liposome-water system) were determined by second-derivative spectrophotometry. The second-derivative spectra of these OPs in the presence of PC SUV showed a bathochromic shift according to the increase in PC concentration and distinct derivative isosbestic points, demonstrating the complete elimination of the residual background signal effects that were observed in the absorption spectra. The Kp values were calculated from the second-derivative intensity change induced by addition of PC SUV and obtained with a good precision of R.S.D. below 10%. The Kp values were in the order of CPFM>FT>PFF>PCF>IFP>CFVP>FNT⩾DZN and did not show a linear correlation relationship with the reported partition coefficients obtained using an n-octanol-water system (R(2)=0.530). Also, the results quantitatively clarified the effect of chemical-group substitution in OPs on their lipophilicity. Since the partition coefficient for the liposome-water system is more effective for modeling the quantitative structure-activity relationship than that for the n-octanol-water system, the obtained results are toxicologically important for estimating the accumulation of these OPs in human cell membranes.

Effect of long-chain Fatty acids on the binding of triflupromazine to human serum albumin: a spectrophotometric study.

Human serum albumin (HSA) in the blood binds long-chain fatty acids (LCFAs), and the number of bound LCFAs varies from 1 to 7 depending on the physical condition of the body. In this study, the influence of LCFA-HSA binding on drug-HSA binding was studied using triflupromazine (TFZ), a psychotropic phenothiazine drug, in a buffer (0.1 M NaCl, pH 7.40, 37°C) by a second-derivative spectrophotometric method which can suppress the residual background signal effects of HSA observed in the absorption spectra. The examined LCFAs were caprylic acid (CPA), lauric acid (LRA), oleic acid (OLA), and linoleic acid (LNA), respectively. Using the derivative intensity change of TFZ induced by the addition of HSA containing LCFA, the binding mode of TFZ was predicted to be a partition-like nonspecific binding. The binding constant (K M(-1)) showed an increase according to the LCFA content in HSA for LRA, OLA, and LNA up to an LCFA/HSA molar ratio of 3-4. However, at higher ratios the K value decreased, i.e. for OLA and LNA, at an LCFA/HSA ratio of 6-7, the K value decreased to 40% of the value for HSA alone. In contrast, CPA, having the shortest chain length (8 carbons) among the studied LCFAs, induced a 20% decrease in the K value regardless of its content in HSA. Since the pharmacological activity of a drug is closely related to the unbound drug concentration in the blood, the results of the present study are pharmaco-kinetically, pharmacologically, and clinically very important.

Characterization, in vitro cytotoxicity and cellular accumulation of paclitaxel-loaded lipid nano-emulsions.

Lipid nano-emulsions (LNEs) having a mean droplet size of approximately 50 nm were investigated as drug carriers for paclitaxel (TXL) to achieve its satisfactory loadings and to develop a pharmaceutically acceptable alternative to the current formulation, Taxol. TXL was incorporated into the LNEs at 2.0 mg/ml without changes in particle size or drug precipitation. In the cytotoxicity study, TXL-loaded LNEs had cytotoxicity to HeLa cells equivalent to that of TXL alone; the 50% growth inhibitory concentrations (IC(50)) of TXL-loaded LNEs and TXL alone were 1.53 +/- 0.23 nM and 1.76 +/- 0.08 nM, respectively. However, a cellular accumulation study using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a fluorescent probe showed that the accumulation of DPH-loaded LNEs in HeLa cells was remarkably lower than that of DPH alone. These results indicated that LNEs were a useful vehicle for TXL, even though LNEs themselves could not be efficiently accumulated in HeLa cells.

Preparation and characterization of a new lipid nano-emulsion containing two cosurfactants, sodium palmitate for droplet size reduction and sucrose palmitate for stability enhancement.

A new lipid nano-emulsion (LNE) was prepared from soybean oil and phosphatidylcholine (PC) employing two cosurfactants, sodium palmitate (PA) for reduced droplet size and sucrose palmitate (SP) for stability enhancement. The mean droplet size of LNEs prepared at a PA/PC (w/w) ratio of larger than 1/10 was found to be ca. 50 nm by dynamic light scattering and atomic force microscopy. However, during the 12-month storage, the PA/PC (1/10)-LNE showed an increase in mean droplet size and broadening of the droplet size distribution due to coalescence of the LNE particles. In a saline solution, the coalescence proceeded very rapidly, i.e., the mean droplet size increased to more than 150 nm within 0.5 h. To suppress the coalescence of LNE particles, four sucrose fatty acid esters of different chain lengths were examined as candidate cosurfactants. The results showed that PA/SP/PC (1/4/10)-LNE could maintain a mean droplet size around 50 nm for 12 months. In a saline solution, the mean droplet size could be maintained within 100 nm even after 24 h. Slight formation of flocculation in the LNEs depending on the storage period was suggested by measurement of the 31P nuclear magnetic resonance line width of the LNEs.

Partitioning of anti-inflammatory steroid drugs into phosphatidylcholine and phosphatidylcholine-cholesterol small unilamellar vesicles as studied by second-derivative spectrophotometry.

The partition coefficients (Kps) of six anti-inflammatory steroid drugs, dexamethasone (DMS), betamethasone (BMS), triamcinolone acetonide (TCLA), fluocinolone acetonide (FCLA), betamethasone 17,21-dipropionate (BMSDP), and clobetasole propionate (CBSP), for phosphatidylcholine (PC), and PC-cholesterol small unilamellar vesicles (SUVs) were determined by a second-derivative spectrophotometric method. The Kp values were obtained with a relative standard deviation of below 10% and the following order was observed: BMS< or =DMS

(19)F NMR spectroscopic characterization of the interaction of niflumic acid with human serum albumin.

The interaction of a non-steroidal anti-inflammatory drug, niflumic acid (NFA), with human serum albumin (HSA) has been investigated by (19)F nuclear magnetic resonance (NMR) spectroscopy. A (19)F NMR spectrum of NFA in a buffered (pH 7.4) solution of NaCl (0.1 mol L(-1)) contained a single sharp signal of its CF(3) group 14.33 ppm from the internal reference 2,2,2-trifluoroethanol. Addition of 0.6 mmol L(-1) HSA to the NFA buffer solution caused splitting of the CF(3) signal into two broadened signals, shifted to the lower fields of 14.56 and 15.06 ppm, with an approximate intensity ratio of 1:3. Denaturation of HSA by addition of 3.0 mol L(-1) guanidine hydrochloride (GU) restored a single sharp signal of CF(3) at 14.38 ppm, indicating complete liberation of NFA from HSA as a result of its denaturation. These results suggest that the binding is reversible and occurs in at least two HSA regions. Competitive (19)F NMR experiments using warfarin, dansyl-L: -asparagine, and benzocaine (site I ligands), and L: -tryptophan and ibuprofen (site II ligands) revealed that NFA binds to site I at two different regions, Ia and Ib, in the ratio 1:3. By use of (19)F NMR with NFA as an (19)F NMR probe the nonfluorinated site I-binding drugs sulfobromophthalein and iophenoxic acid were also found to bind sites Ia and Ib, respectively. These results illustrate the usefulness and convenience of (19)F NMR for investigation of the HSA binding of both fluorinated and nonfluorinated drugs.

Effects of inorganic ions on the binding of triflupromazine and chlorpromazine to bovine serum albumin studied by spectrometric methods.

The effects of inorganic salts, NaCl, NaBr, NaI, Na2SO4, KCl, KBr, KI, on the binding constants (Ks) of psychotropic phenothiazine drugs, triflupromazine (TFZ) and chlorpromazine, to bovine serum albumin (BSA) were examined by using second-derivative spectrophotometry. All of the salts examined, with the exception of Na2SO4, decreased the K values significantly, depending on the concentration of the salt, e.g., the decrease in the K values of both drugs were about 40% for 0.1 M NaCl. The results obtained with Na2SO4 indicated that neither Na+ nor SO4(2-) had any affect on the binding of the phenothiazines to BSA. Based on the Na2SO4 results and the finding that the effect of each potassium salt on binding was quite similar to that of the corresponding sodium salt, the effects of these halogen salts can be considered to be derived from their anions, although the phenothiazines are positively charged at pH 7.4. The effectiveness of the anions was determined to occur in the following order: I->>Br->Cl-; these results coincided with the published order of the binding affinity of these anions to albumin. The 19F-NMR spectra of TFZ in the presence of each of these halogen salts revealed a concentration-dependent decrease in the intensity of the signal at 13.8 ppm that had previously been assigned to the TFZ bound to Site II. Consequently, the effects of these anions on the binding of positively charged phenothiazine drugs are thought to be local steric effects caused by the binding of these anions to Site II.

Needle-type ultra micro silver/silver chloride reference electrode for use in micro-electrochemistry.

A needle-type ultra micro silver/silver chloride reference electrode having a micro capillary with outer and inner diameters of 1.0 microm and 0.5 +/- 0.2 microm, respectively, was constructed. This micro reference electrode can be stuck into a living cell, and is applicable to use in very small environments, such as an electrochemical cell of an electrochemical scanning tunneling microscope or the detection portion of a micro-TAS. Excellent stability and repeatability of the micro reference electrode potential could be obtained by filling the micro capillary with agar gel containing 3.33 mol/L potassium chloride as a salt bridge, by covering the bare part of the silver wire on which silver chloride was deposited, and by electromagnetic shielding of the measurement cell and wire lead from the electromagnetic waves. The electrode showed stable potential for 7 days after its fabrication using 3.3 mol/L potassium chloride aqueous solution containing silver chloride as an internal electrolyte solution. The electrode exhibited constant electrode potential and excellent stability in test solutions of pH 5-9. The electrode potential of a commercial pH glass electrode measured against the micro reference electrode in standard pH buffer solutions was in good accordance with the Nernst equation.

Effects of phosphatidylserine and phosphatidylethanolamine content on partitioning of triflupromazine and chlorpromazine between phosphatidylcholine-aminophospholipid bilayer vesicles and water studied by second-derivative spectrophotometry.

To assess the affinity of psychotropic phenothiazine drugs, triflupromazine (TFZ) and chlorpromazine (CPZ), for the membranes of central nervous system and the other organs in the body, the partition coefficients (Kps) of these drugs to phosphatidylcholine (PC)-phosphatidylserine (PS) and PC-phosphatidylethanolamine (PE) small and large unilamellar vesicles (SUV, LUV) were examined by a second-derivative spectrophotometric method, since PS is abundantly contained in the membranes of the central nervous system and PE is distributed widely in the membranes of the organs in the body. Size and preparation methods of the vesicles did not affect the Kp values at each aminophospholipid content suggesting that the partition of the phenothiazine drugs was not affected by the structural differences in the vesicles such as their curvature or asymmetric distribution of the phospholipids between the outer and inner layers of the bilayer membranes. However, the Kp values of both drugs increased remarkably according to the PS content in the bilayer membranes, i.e., the Kp values for the vesicles of 30 mol% PS content were about 3 times of that for the vesicles of PC alone, while both Kp values slightly reduced with the increase in the content of PE in the bilayer membranes of PC-PE vesicles. The results indicate that both drugs have higher affinity for the PC-PS bilayer membranes than for the PC and PC-PE membranes, which can offer an evidence for the fact that TFZ and CPZ are predominantly distributed and accumulated in the brain and nerve cell membranes that contain PS abundantly.

Potentiometric immunosensor using artificial antibody based on molecularly imprinted polymers.

A potentiometric artificial immunosensor based on a molecularly imprinted polymer was prepared as a detecting element in micro total analysis systems with the intent of providing easy clinical analysis. As the structure and transducing mechanism of this sensor are very simple, construction of a single microsensor should be quite easy. Multimicrosensor arrays applicable to several kinds of analytes will be attainable by both changing the template molecule to be imprinted and reducing the sensor size. The response characteristics of this sensor were evaluated by measuring the response potential to serotonin, which was used as a model material. The obtained sensor was highly responsive to serotonin in water but not to tryptamine, acetaminophen, or procainamide. This phenomenon confirms that the sensor recognizes serotonin and that it functions as a specific artificial immunosensor. Quick measurement is possible because the response time, defined as the time required to achieve 95% of the magnitude of the equilibrated signal, correspond to approximately 12 s. The sensor's determination and detection limits were found to be 1 mumol/L and 100 pmol/L, respectively. These results suggest that our strategy can be applied to construction of a potentiometric artificial immunosensor.