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

• Published in: Journal of Chromatography A Vol. 1217; no. 36; pp. 5732 - 5736
• Main Authors: Zhao, Shulin, Huang, Yong, Ye, Fanggui, Shi, Ming, Liu, Yi-Ming
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 03.09.2010; Amsterdam; New York: Elsevier; Elsevier
• Subjects: Analytical biochemistry: general aspects, technics, instrumentation; Analytical, structural and metabolic biochemistry; biogenic amines; Biological and medical sciences; chemiluminescence; Chemiluminescence detection; chromatography; cysteine; Cysteine - analysis; cytolysis; detection limit; electrophoresis; Electrophoresis, Microchip - methods; erythrocytes; Erythrocytes - chemistry; Fundamental and applied biological sciences. Psychology; glutathione; Glutathione - analysis; hemoglobin; Hemoglobins - analysis; Humans; Investigative techniques, diagnostic techniques (general aspects); Linear Models; Liver Neoplasms; Luminescent Measurements - methods; Medical sciences; Microchip electrophoresis; Miscellaneous. Technology; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; patients; Reproducibility of Results; Sensitivity and Specificity; Single cell analysis; Sulfhydryl Compounds - analysis; Sulphydryl compounds; Thyroid Neoplasms; Sulphydryl compounds; Microchip electrophoresis; Chemiluminescence detection; Single cell analysis; Cysteine; Hemoprotein; Chemical analysis; Tripeptide; Hemoglobin; Quantitative analysis; Glutathione; Human; Fluid mechanics; Thiol; Capillary electrophoresis; Red blood cell; Single cell; Patient; Chemiluminescence; System on a chip; Malignant tumor; Normal; Protein; Sulfur containing aminoacid; Sulfur peptide; Intracellular; Miniaturization; Microfluidics; Cancer
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2010.07.012

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–Na 2S 2O 8 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.