Development of a gold nanoparticles based chemiluminescence imaging assay and its application

In this paper, a novel gold nanoparticles based protein immobilization method was designed. Biocomposites of gold nanoparticles and proteins were successfully coated on poly(methyl methacrylate) (PMMA) plates and polystyrene microtiter plates. The proteins could be immobilized on solid materials with high density and better bioactivity. Based on above design, chemiluminescence (CL) imaging assay for determination of H₂O₂ and recombinant human interleukin-6 (rHu IL-6) was developed. The linear range and the loading capability were greatly improved when compared with imaging assay performed with direct proteins immobilization. Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of H₂O₂ in the range of 1.0 × 10⁻⁶ to 1.0 × 10⁻⁴ mol L⁻¹, and rHu IL-6 in the range of 2.0-312.0 pg mL⁻¹. The detection limits were 2 × 10⁻⁷ mol L⁻¹ (3σ) for H₂O₂ and 0.5 pg mL⁻¹ for rHu IL-6 with relative standard deviation of 3.8% for 3.0 × 10⁻⁵ mol L⁻¹ H₂O₂, and 4.4% for 39.0 pg mL⁻¹ rHu IL-6. This method has been applied to the determination of rHu IL-6 in human serum with satisfactory results.     

Determination of beta-lactam antibiotics in milk using micro-flow chemiluminescence system with on-line solid phase extraction

In this paper, a chemiluminescence (CL) micro-flow system combined with on-line solid phase extraction (SPE) is presented for determination of β-lactam antibiotics (penicillin, cefradine, cefadroxil, cefalexin) in milk. It is based on the enhancement effect of β-lactam antibiotics on the luminol-K₃Fe(CN)₆ CL system. The micro-flow system was fabricated from two polymethyl methacrylate (PMMA) plates (50 mm × 40 mm × 5 mm) with the microchannels of 200 μm wide and 150 μm deep. C₁₈-modified silica gel was packed into the microchannel (length: 10 mm; width: 1 mm; depth: 500 μm) to serve as SPE device. Extraction and preconcentration of the analytes were carried out using on-line SPE micro-flow system and the selectivity of CL detection was improved. The detection limits were 0.5 μg mL⁻¹ of penicillin, 0.04 μg mL⁻¹ of cefradine, 0.08 μg mL⁻¹ of cefadroxil and 0.1 μg mL⁻¹ of cefalexin. The proposed method was also applied to analyze the β-lactam antibiotics in milk. Experimental results were in good agreement with those obtained by high performance liquid chromatography (HPLC) method with UV detection.     

Chemiluminescence flow-through sensor for pipemidic acid using solid sodium bismuthate as an oxidant

A novel chemiluminscence (CL) flow-through sensor for pipemidic acid is described. It was based on the sensitizing effect of pipemidic acid on the CL oxidation of sulfite by sodium bismuthate in H₂SO₄ media. The solid-phase sodium bismuthate was mechanicially immobilized on the sponge rubber inside of the CL flow cell as CL oxidant. The calibration graph is linear in the range 0.1-10 μg/ml with a detection limit of 6.2×10⁻⁸ g/ml (3σ). A complete analysis could be performed in 1 min with a relative standard deviation (R.S.D.) of 2.5% for 2 μg/ml pipemidic acid (n=8). This method has been successfully applied to determine pipemidic acid in pharmaceutical preparation.     

Measurement of bisphenol A in human serum by gas chromatography/mass spectrometry

The purpose of this study is to establish an easy and accurate method for the determination of bisphenol A (BPA) in the human serum. The samples were applied to the C₁₈ solid phase extraction (SPE) column for clean up of samples. The BPA is conjugated with tetrabutylammonium hydrogen sulfate as the counter ion in alkali solution. The ion paired BPA is moves from the aqueous phase to the organic phase as an ion paired extraction. BPA extracted from human serum were derivatized with pentafluorobenzyl bromide (PFBBr). The derivative was analyzed by gas chromatography (GC)/mass spectrometry (MS) using negative chemical ionization (NCI). The instrumental detection limit of BPA was 5 pg/ml (10 fg). The instrumental response between 0.01 and 100 pg/ml of BPA standards was linear (r²=0.998). The recovery of BPA spiked into human serum was 101.0±4.63 (1 pg/ml) and 100.9±3.75 (10 pg/ml), respectively. The concentration of BPA in the human serum from 20 individuals was 0.54 pg/ml.     

Highly sensitive multianalyte immunochromatographic test strip for rapid chemiluminescent detection of ractopamine and salbutamol

A novel immunochromatographic assay (ICA) was proposed for rapid and multiple assay of β₂-agonists, by utilizing ractopamine (RAC) and salbutamol (SAL) as the models. Owing to the introduction of chemiluminescent (CL) approach, the proposed protocol shows much higher sensitivity. In this work, the described ICA was based on a competitive format, and horseradish peroxidase-tagged antibodies were used as highly sensitive CL probes. Quantitative analysis of β₂-agonists was achieved by recording the CL signals of the probes captured on the two test zones of the nitrocellulose membrane. Under the optimum conditions, RAC and SAL could be detected within the linear ranges of 0.50–40 and 0.10–50 ng mL⁻¹, with the detection limits of 0.20 and 0.040 ng mL⁻¹ (S/N = 3), respectively. The whole process for multianalyte immunoassay of RAC and SAL can be completed within 20 min. Furthermore, the test strip was validated with spiked swine urine samples and the results showed that this method was reliable in measuring β₂-agonists in swine urine. This CL-based multianalyte test strip shows a series of advantages such as high sensitivity, ideal selectivity, simple manipulation, high assay efficiency and low cost. Thus, it opens up new pathway for rapid screening and field analysis, and shows a promising prospect in food safety.     

Polymer nanoparticles as fluorescent labels in a fluoroimmunoassay for human chorionic gonadotropin

A sensitive fluoroimmunoassay (FIA) was developed for the determination of human chorionic gonadotropin (β-HCG). It is based on fluorescent polymer nanoparticles (PFNPs) coated with anti-β-HCG monoclonal antibodies in a sandwich type of fluoroimmunoassay. The PFNPs were synthesized by precipitation polymerization using methacrylic acid (MAA) as the monomer, trimethylolpropane trimethacrylate as the cross-linker, azobisisobutyronitrile as the radical initiator, and fluorescein as the fluorophore. Anti-β-HCG monoclonal antibody was labeled with the PFNPs and then used in a FIA of β-HCG in human serum samples using low-fluorescent transparent 96-well microtiter plates. The calibration graph for β-HCG is linear over the range from 1.25 to 300 mIU mL-¹ with a detection limit of 0.3 mIU mL-¹ (3σ). The relative standard deviation for seven parallel measurements of 10 mIU mL-¹ of β-HCG is 3.8%. The method has the specificity of an immunoassay and the sensitivity of fluorescent nanoparticle label technology.     

Detection of paracetamol by capillary electrophoresis with chemiluminescence detection

Indirect detection of paracetamol was accomplished using a capillary electrophoresis-chemiluminescence (CE-CL) detection system, which was based on its inhibitory effect on a luminol-potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) CL reaction. Paracetamol migrated in the separation capillary, where it mixed with luminol included in the running buffer. The separation capillary outlet was inserted into the reaction capillary to reach the detection window. A four-way plexiglass joint held the separation capillary and the reaction capillary in place. K₃[Fe(CN)₆] solution was siphoned into a tee and flowed down to the detection window. CL was observed at the tip of the separation capillary outlet. The CL reaction of K₃[Fe(CN)₆] oxidized luminol was employed to provide the high and constant background. Since paracetamol inhibits the CL reaction, an inverted paracetamol peak can be detected, and the degree of CL suppression is proportional to the paracetamol concentration. Maximum CL signal was observed with an electrophoretic buffer of 30 mM sodium borate (pH 9.4) containing 0.5 mM luminol and an oxidizer solution of 0.8 mM K₃[Fe(CN)₆] in 100 mM NaOH solution. Under the optimal conditions, a linear range from 6.6 × 10⁻¹⁰ to 6.6 × 10⁻⁸ M (r = 0.9999), and a detection limit of 5.6 × 10⁻¹⁰ M (signal-to-noise ratio = 3) for paracetamol were achieved. The relative standard deviation (R.S.D.) of the peak area for 5.0 × 10⁻⁹ M of paracetamol (n = 11) was 2.9%. The applicability of the method for the analysis of pharmaceutical and biological samples was examined.     

Determination of levodopa by capillary electrophoresis with chemiluminescence detection

A rapid and simple method using capillary electrophoresis (CE) with chemiluminescence (CL) detection was developed for the determination of levodopa. This method was based on enhance effect of levodopa on the CL reaction between luminol and potassium hexacyanoferrate(III) (K₃[Fe(CN)₆]) in alkaline aqueous solution. CL detection employed a lab-built reaction flow cell and a photon counter. The optimized conditions for the CL detection were 1.0 × 10⁻⁵ M luminol added to the CE running buffer and 5.0 × 10⁻⁵ M K₃[Fe(CN)₆] in 0.6 M NaOH solution introduced postcolumn. Under the optimal conditions, a linear range from 5.0 × 10⁻⁸ to 2.5 × 10⁻⁶ M (r = 9991), and a detection limit of 2.0 × 10⁻⁸ M (signal/noise = 3) for levodopa were achieved. The precision (R.S.D.) on peak area (at 5.0 × 10⁻⁷ M of levodopa, n = 11) was 4.1%. The applicability of the method for the analysis of pharmaceutical and human plasma samples was examined.     

A novel gaseous pinacolyl alcohol sensor utilizing cataluminescence on alumina nanowires prepared by supercritical fluid drying

A cataluminescence (CTL) sensor using Al₂O₃ nanowires as the sensing material was developed for the determination of trace pinacolyl alcohol in air samples based on the catalytic chemiluminescence (CL) of pinacolyl alcohol on Al₂O₃ nanowires. Eight catalysts were examined and the CL intensity on Al₂O₃ nanowires prepared by supercritical fluid drying was the strongest. This novel CL sensor showed high sensitivity and selectivity to gaseous pinacolyl alcohol at optimal temperature of 340 °C. Quantitative analysis was performed at a wavelength of 460 nm. The linear range of CTL intensity versus concentration of gaseous pinacolyl alcohol was 0.09 × 10⁻⁶ to 2.56 × 10⁻⁶ g mL⁻¹ (r = 0.9983, n = 6) with a detection limit (3σ) of 0.0053 × 10⁻⁶ g mL⁻¹. None or only very low levels of interference were observed while the foreign substances such as water vapor, ethanol, ammonia, chloroform, benzene, nitrogen dioxide, methylbenzene, hydrochloric acid, methanol and butanol were passing through the sensor. The response time of the sensor is less than 100 s, and the sensor had a long lifetime more than 60 h. The sensor would be potentially applied to analysis of the nerve agents such as Soman.     

Flow injection chemiluminescence determination of naproxen based on KMnO4-Na2SO3 reaction in neutral aqueous medium

A simple, rapid and sensitive flow injection chemiluminescence (CL) method is described for the determination of naproxen. It was found that strong CL signal was generated when naproxen was mixed with KMnO₄ and Na₂SO₃ in neutral aqueous medium. Under the optimum experimental conditions, the CL intensity was linearly related to the concentration of naproxen from 4.0 × 10⁻⁹ to 1.0 × 10⁻⁶ g mL⁻¹ (r = 0.9993). The detection limit was 2 × 10⁻⁹ g mL⁻¹ naproxen, the relative standard deviation for 1.0 × 10⁻⁷ g mL⁻¹ naproxen solution was 1.5% (n = 11) and the sampling frequency was 120 h⁻¹. The method was applied to the determination of naproxen in pharmaceutical preparation with satisfactory results. The mechanism of CL reaction was discussed briefly.     

Flow injection chemiluminescence determination of nitrofurazone in pharmaceutical preparations and biological fluids based on oxidation by singlet oxygen generated in N-bromosuccinimide-hydrogen peroxide reaction

A simple flow injection chemiluminescence (FI-CL) method was proposed for the determination of nitrofurazone. Strong CL signal was generated during the reaction of nitrofurazone with H₂O₂ and N-bromosuccinimide (NBS) in alkaline condition. The CL signal was proportional to the nitrofurazone concentration in the range 1.0 × 10⁻⁷ to 1.0 × 10⁻⁵ g mL⁻¹. The detection limit was 2 × 10⁻⁸ g mL⁻¹ nitrofurazone and the relative standard deviation was less than 4% (6.0 × 10⁻⁶ g mL⁻¹ nitrofurazone, n = 11). The proposed method was successfully applied to the determination of nitrofurazone in compound furacillin nasal drops, human plasma and urine samples. The CL reaction mechanism was also discussed briefly. Singlet oxygen generated in the reaction between H₂O₂ and NBS was suggested to be participated in the CL reaction.     

Investigation on enhanced chemiluminescence reaction systems with bis(hydrogenperiodato) argentate(III) complex anion for fluoroquinolones synthetic antibiotics

A novel chemiluminescence (CL) reaction system with bis(hydrogenperiodato) argentate(III) complex anion (Ag(III) complex, [Ag(HIO₆)₂]⁵⁻), for the first time, is developed for the determination of lomefloxacin (LMFX), enrofloxacin (ENLX) and pefloxacin (PFLX). The possible CL emission mechanism was discussed by comparing the fluorescence emission with CL spectra. The CL conditions of [Ag(HIO₆)₂]⁵⁻-H₂SO₄-LMFX/ENLX/PFLX systems were investigated and optimized. Under the optimized experimental conditions, the CL intensity is proportional to the concentration of the drugs in the range 0.2994-36.80 × 10⁻⁷ g mL⁻¹ for LMFX, 4.00-30.0 × 10⁻⁷ g mL⁻¹ for ENLX and 1.54-27.64 × 10⁻⁷ g mL⁻¹ for PFLX. The limit of detection (s/n = 3) was 9.1 × 10⁻⁹ g mL⁻¹ for LMFX, 3.1 × 10⁻⁹ g mL⁻¹ for ENLX and 4.4 × 10⁻⁹ g mL⁻¹ for PFLX. The recovery of LMFX, ENLX and PELX from the spiked pharmaceutical preparations was in the range of 92.3-105% with the RSDs of 0.5-2.7%. For urine, serum and milk samples the recoveries of the three drugs were in the range of 85.1-107% for LMFX with the RSDs of 2.3-3.4%. 80.2-112% for ENLX with the RSDs of 1.4-2.8%, and 87.8-114% for PFLX with the RSDs of 1.6-2.7%. The proposed method was applied successfully to the determination of these compounds in real samples.     

Investigation on sensitized chemiluminescence systems and their mechanism for five fluoroquinolones

The novel chemiluminescence (CL) reaction systems were established for lomefloxacin (LMFX), ofloxacin(OFLX), norfloxacin (NFLX), gatifloxacin (GAFX) and enoxacin (ENX). The sensitized CL emission mechanism was investigated for the five systems by comparing the fluorescence emission with CL spectra. For LMFX-Ce(IV)-S₂O₃²⁻-H₂SO₄ and OFLX-Ce(IV)-S₂O₄²⁻-H₂SO₄ systems, the CL intensity is enhanced through intermolecular energy transfer from the excited SO₂^* to LMFX and OFLX. For NFLX-Ce(IV)-S₂O₄²⁻-HNO₃ system, the sensitized CL is based on intermolecular energy transfer from the excited SO₂^* to NFLX oxide. For Eu³⁺-GAFX-Ce(IV)-S₂O₄²⁻-HCl and Dy³⁺-ENX-Ce(IV)-S₂O₃²⁻-H₂SO₄ systems, the CL spectra are from the narrow characteristic emission at 590, 619 and 649 nm of Eu^3+* (⁵D₀ → ⁷F₁, ⁵D₀ → ⁷F₂, ⁵D₀ → ⁷F₃) and at 482 and 578 nm of Dy³⁺ (⁴F₉ → ⁶H_15/2, ⁴F₉ → ⁶H_13/2) through intermolecular energy transfer from the excited SO₂^* to GAFX and ENX, followed by intramolecular energy transfer from GAFX^* to Eu³⁺ and ENX^* to Dy³⁺. The conditions of CL emission were investigated and optimized. The proposed five enhanced CL systems have good linearity, higher sensitivity, precision and potential capability for residue analysis of studied analytes in foods and biological samples.     

Application of a novel co-enzyme reactor in chemiluminescence flow-through biosensor for determination of lactose

A novel enzyme reactor with co-immobilization of β-galactosidase and glucose oxidase in calcium alginate fiber (CAF) and amine modified nanosized mesoporous silica (AMNMS) was prepared which incorporate the adsorption and catalysis of AMNMS with the cage effect of the polymer to increase catalytic activity and stability of immobilized enzyme. The enzyme reactor was applied to prepare a chemiluminescence (CL) flow-through biosensor for determination of lactose combined with a novel luminol-diperiodatonickelate (DPN) CL system we reported. It shows that the CL flow-through biosensor possesses long lifetime, high stability, high catalytic activity and sensitivity. The relative CL intensity was linear with the lactose concentration in the range of 8 × 10⁻⁸-4 × 10⁻⁶ g mL⁻¹ with the detection limit of 2.7 × 10⁻⁸ g mL⁻¹ (3σ). It has been successfully applied to the determination of lactose in milk.     

Determination of intracellular sulphydryl compounds by microchip electrophoresis with selective chemiluminescence detection

An analytical method based on microchip electrophoresis (MCE) and chemiluminescence detection (CL) was developed for the determination of intracellular sulphydryl compounds. Cell injection/loading, cytolysis, electrophoretic separation, and CL detection were integrated onto a simple cross-microfluidic chip. Selective CL detection of sulphydryl compounds was achieved by deploying the luminol–Na₂S₂O₈ reaction. Under the CL conditions selected, many endogenous compounds in biological systems such as amino acids, biogenic amines, peptides and proteins did not produce any CL signal, which further ensured a high selectivity of the proposed MCE–CL assays. Sulphydryl compounds including cysteine (Cys), glutathione (GSH), and hemoglobin (Hb) were selected as the test compounds. The MCE separation was completed within 120 s. The detection limits were estimated to be 7 amol for Cys, 32 amol for GSH and 69 amol for Hb, respectively. The present method was applied to analyze individual red blood cells collected from both healthy subjects and cancer patients. It was found that the average intracellular contents of Cys, GSH and Hb were in the ranges of 26–43 amol/cell, 128–323 amol/cell and 522–667 amol/cell, respectively for cancer patients, compared to 579–609 amol Hb/cell and not detectable Cys and GSH for healthy subjects.     

Determination of bisphenol A based on chemiluminescence from gold(III)-peroxymonocarbonate

Peroxymonocarbonate (HCO₄⁻) was produced by the online reaction of bicarbonate with hydrogen peroxide. A strong chemiluminescence (CL) was observed when HCO₄⁻ reacted with AuCl₄⁻ without any special CL reagent. When bisphenol A (BPA) was added to AuCl₄⁻-HCO₄⁻ CL system, the CL emission was inhibited significantly. This new CL system was developed as a flow-injection method for the determination of BPA. Under the optimum experimental conditions, the inhibited CL intensity was linearly related to the concentration of BPA from 0.3 to 80 μM (R = 0.9958). The detection limit of BPA was 0.08 μM. The relative standard deviation for 12 repeated measurements of 1.0 μM BPA was 2.9%. The interferences of some cationic ions can be removed by an online cation-exchange column. The applicability of the present CL system was demonstrated for the sensitive and selective determination of BPA in real samples (mineral water bottle, baby bottle, beverage bottle and polycarbonate container). Based on the CL spectrum, UV-visible adsorption spectra, and the quenching effect of reactive oxygen species scavengers, a possible CL mechanism was proposed.     

β−cyclodextrins-based inclusion complexes of CoFe2O4 magnetic nanoparticles as catalyst for the luminol chemiluminescence system and their applications in hydrogen peroxide detection

β−cyclodextrins (β−CD)-based inclusion complexes of CoFe₂O₄ magnetic nanoparticles (MNPs) were prepared and used as catalysts for chemiluminescence (CL) system using the luminol-hydrogen peroxide CL reaction as a model. The as-prepared inclusion complexes were characterized by XRD (X-ray diffraction), TGA (thermal gravimetric analysis) and FT-IR. The oxidation reaction between luminol and hydrogen peroxide in basic media initiated CL. The effect of β−CD-based inclusion complexes of CoFe₂O₄ magnetic nanoparticles and naked CoFe₂O₄ magnetic nanoparticles on the luminol-hydrogen peroxide CL system was investigated. It was found that inclusion complexes between β−CD and CoFe₂O₄ magnetic nanoparticles could greatly enhance the CL of the luminol-hydrogen peroxide system. Investigation on the kinetic curves and the chemiluminescence spectra of the luminol-hydrogen peroxide system demonstrates that addition of CoFe₂O₄ MNPs or inclusion complexes between β−CD and CoFe₂O₄ MNPs does not produce a new luminophor of the chemiluminescent reaction. The luminophor for the CL system was still the excited-state 3-aminophthalate anions (3-APA*). The enhanced CL signals were thus ascribed to the possible catalysis from CoFe₂O₄ MNPs or inclusion complexes between β−CD and CoFe₂O₄ nanoparticles. The feasibility of employing the proposed system for hydrogen peroxide sensing was also investigated. Experimental results showed that the CL emission intensity was linear with hydrogen peroxide concentration in the range of 1.0 × 10⁻⁷ to 4.0 × 10⁻⁶ mol L⁻¹ with a detection limit of 2.0 × 10⁻⁸ mol L⁻¹ under optimized conditions. The proposed method has been used to determine hydrogen peroxide in water samples successfully.     

Sub-picogram determination of Vitamin B12 in pharmaceuticals and human serum using flow injection with chemiluminescence detection

A sensitive chemiluminescence (CL) method, based on the enhancive effect of cobalt(II) on the CL reaction between luminol and dissolved oxygen in a flow injection (FI) system, was proposed for determination of Vitamin B₁₂. The increment of the CL intensity was proportional to the concentration of Vitamin B₁₂, giving a calibration graph linear over the concentration from 2.0×10⁻¹⁰ to 1.2×10⁻⁶ g l⁻¹ (r²=0.9992) with the detection limit of 5.0×10⁻¹¹ g l⁻¹ (3σ). At a flow rate of 2.0 ml min⁻¹, a complete determination of Vitamin B₁₂, including sampling and washing, could be accomplished in 0.5 min with the relative standard deviations (R.S.D.) of less than 5.0%. The proposed method was applied successfully to the determination of Vitamin B₁₂ in pharmaceuticals, human serum, egg yolk and fish tissue.     

Microfluidic device capable of sensing ultrafast chemiluminescence

Based on the principle of liquid core waveguide, a novel microfluidic device with micro-scale detection window capable of sensing flashlight emitted from rapid 1,1′-oxalyldi-4-methylimidazole (OD4MI) chemiluminescence (CL) reaction was fabricated. Light emitted from OD4MI CL reaction occurring in the micro-dimensional pentagonal detection window (length of each line segment: 900.0 μm, depth: 50.0 μm) of the microfluidic device with two inlets and one outlet was so bright that it was possible to take an image every 1/30 s at the optimal focusing distance (60 cm) using a commercial digital camera. Peaks obtained using a flow injection analysis (FIA) system with the micro-scale detection window and OD4MI CL detection show excellent resolution and reproducibility without any band-broadening observed in analytical devices having additional reaction channel(s) to measure light generated from slow CL reaction. Maximum height (H_max) and area (A) of peak, reproducibility and sensitivity observed in the FIA system with the microfluidic device and OD4MI CL detection depends on (1) the mole ratio between bis(2,4,6-trichlorophenyl) oxalate and 4-methyl imidazole yielding OD4MI, (2) the flow rate to mix OD4MI, H₂O₂ and 1-AP in the detection window of the microfluidic device, and (3) H₂O₂ concentration. We obtained linear calibration curves with wide dynamic ranges using H_max and A. The detection limit of 1-AP determined with H_max and A was as low as 0.05 fmole/injection (signal/background = 3.0).     

Microchip electrophoresis with chemiluminescence detection for assaying ascorbic acid and amino acids in single cells

A method based on microchip electrophoresis (MCE) with chemiluminescence (CL) detection was developed for the determination of ascorbic acid (AA) and amino acids including tryptophan (Trp), glycine (Gly) and alanine (Ala) present in single cells. Cell injection, loading, lysing, electrophoretic separation and CL detection were integrated onto a simple cross microfluidic chip. A single cell was loaded in the cross intersection by electrophoretic means through applying a set of potentials at the reservoirs. The docked cell was lysed rapidly under a direct electric field. The intracellular contents were MCE separated within 130 s. CL detection was based on the enhancing effects of AA and amino acids on the CL reaction of luminol with K₃[Fe(CN)₆]. Rat hepatocytes were prepared and analyzed as the test cellular model. The average intracellular contents of AA, Trp, Gly and Ala in single rat hepatocytes were found to be 38.3, 5.15, 3.78 and 3.84 fmol (n = 12), respectively.