Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser‐induced fluorescence detection

Abstract

A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 microm erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser-induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8-32 amol/single cell.

Importance of the counterion in optimization of a borate electrolyte system for analyses of anions in samples with complex matrices performed by capillary zone electrophoresis

“…For the pH range 8.7-9.7 this electrolyte system acts as a good buffer for CZE analyses. Its application field ranges from determination of small ions to large molecules both in simple matrices, such as pharmaceutical mixtures and in the most complex fluids, serum, and urine [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17], and it is also favored in chiral separations or micellar electrokinetic chromatography (MEKC) with cyclodextrins [18][19][20][21][22][23][24][25], sodium dodecyl sulfate [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42], or other surfactants [43,44] added, as well as in microemulsion electrokinetic chromatography (MEEKC) [45]. As an electrolyte system used for the separation of anions, it is the borate co-ion that predominantly determines the adjustation of concentration of the anionic analytes in their zones ...…”

Intracellular FITC-derivatization with PEG

“…Thus, it is important to develop a simple and cheap method for fluorescent markers of AAs in plant cells. To do this, we need to minimize the cell dilution during derivatization, so as to maintain favorable kinetics for the labeling reaction, as well as to maintain AA concentrations is already present at the trace level [1,2]. A derivatization method in single-cell analysis by open-tubular (OT) LC was described by Jorgenson et al [3,4].…”

“…100) [5]. Zhang and Jin [1] developed a method of intracellular FITC-derivatization by electroporation, with a dilution factor reduced from ca. 100 to ca.…”

“…The derivatized AAs released from the cells were separated by CE and detected by a sensitive LIF. There were some reports in the area of AA analysis by CE, using electrochemical (EC) or LIF detection [1,2,[10][11][12][13]. A P/ACE MDQ CE system (Beckman Instruments, Fullerton, CA, USA) equipped with an LIF detector (488 nm laser module, Beckman Coulter) was used to perform all separations and detections in this study.…”

“…After sufficient derivatization for 40 h in the dark [1], the cells were lysed immediately by 0.1 mol/L NaOH, after the lysing solution was added into the mixture containing FITC-derivatized cells. The derivatized AAs released from the cells were separated by CE and detected by a sensitive LIF.…”