Dechen Jiang

Potential-Resolved Electrochemiluminescence for Determination of Two Antigens at the Cell Surface

The potential-resolved electrochemiluminescence (ECL) was achieved for the determination of two antigens at the cell surface through a potential scanning on the electrode. Luminol and Ru(bpy)3(2+) groups as ECL probes were linked with the antibodies to recognize the corresponding antigens on the cell surface. A self-quenching of luminescence from the luminol group under negative potential was initialized by the introduction of concentrated aqueous luminol, which offered accurate measurements of the luminescence from luminol and Ru(bpy)3(2+) groups under positive and negative potentials, respectively. Using this strategy, carcinoembryonic (CEA) and alphafetoprotein (AFP) antigens on cells as the models were quantified serially through a potential scanning. Different patterns of luminescence were observed at MCF 7 and PC 3 cells, which exhibited that the assay can characterize the cells with a difference expression of antigens. Compared with fluorescence measurement, the potential resolved ECL for the detection of two analytes was not limited by the spectrum difference of probes. The strategy involving potential-induced signals required a simplified optical setup and eventually offered an alternative imaging method for multiply antigens in immunohistochemistry.

Luminol electrochemiluminescence for the analysis of active cholesterol at the plasma membrane in single mammalian cells.

A luminol electrochemiluminescence assay was reported to analyze active cholesterol at the plasma membrane in single mammalian cells. The cellular membrane cholesterol was activated by the exposure of the cells to low ionic strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT). The active membrane cholesterol was reacted with cholesterol oxidase in the solution to generate a peak concentration of hydrogen peroxide on the electrode surface, which induced a measurable luminol electrochemiluminescence. Further treatment of the active cells with mevastatin decreased the active membrane cholesterol resulting in a drop in luminance. No change in the intracellular calcium was observed in the presence of luminol and voltage, which indicated that our analysis process might not interrupt the intracellular cholesterol trafficking. Single cell analysis was performed by placing a pinhole below the electrode so that only one cell was exposed to the photomultiplier tube (PMT). Twelve single cells were analyzed individually, and a large deviation on luminance ratio observed exhibited the cell heterogeneity on the active membrane cholesterol. The smaller deviation on ACAT/HMGCoA inhibited cells than ACAT inhibited cells suggested different inhibition efficiency for sandoz 58035 and mevastatin. The new information obtained from single cell analysis might provide a new insight on the study of intracellular cholesterol trafficking.

Analysis of Intracellular Glucose at Single Cells Using Electrochemiluminescence Imaging

Here, luminol electrochemiluminescence was first applied to analyze intracellular molecules, such as glucose, at single cells. The individual cells were retained in cell-sized microwells on a gold coated indium tin oxide (ITO) slide, which were treated with luminol, triton X-100, and glucose oxidase simultaneously. The broken cellular membrane in the presence of triton X-100 released intracellular glucose into the microwell and reacted with glucose oxidase to generate hydrogen peroxide, which induced luminol luminescence under positive potential. To achieve fast analysis, the luminescences from 64 individual cells on one ITO slide were imaged in 60 s using a charge-coupled device (CCD). More luminescence was observed at all the microwells after the introduction of triton X-100 and glucose oxidase suggested that intracellular glucose was detected at single cells. The starvation of cells to decrease intracellular glucose produced less luminescence, which confirmed that our luminescence intensity was correlated with the concentration of intracellular glucose. Large deviations in glucose concentration at observed single cells revealed high cellular heterogeneity in intracellular glucose for the first time. This developed electrochemiluminescence assay will be potentially applied for fast analysis of more intracellular molecules in single cells to elucidate cellular heterogeneity.

Electrochemiluminescence Imaging for Parallel Single-Cell Analysis of Active Membrane Cholesterol

Luminol electrochemiluminescence (ECL) imaging was developed for the parallel measurement of active membrane cholesterol at single living cells, thus establishing a novel electrochemical detection technique for single cells with high analysis throughput and low detection limit. In our strategy, the luminescence generated from luminol and hydrogen peroxide upon the potential was recorded in one image so that hydrogen peroxide at the surface of multiple cells could be simultaneously analyzed. Compared with the classic microelectrode array for the parallel single-cell analysis, the plat electrode only was needed in our ECL imaging, avoiding the complexity of electrode fabrication. The optimized ECL imaging system showed that hydrogen peroxide as low as 10 μM was visible and the efflux of hydrogen peroxide from cells could be determined. Coupled with the reaction between active membrane cholesterol and cholesterol oxidase to generate hydrogen peroxide, active membrane cholesterol at cells on the electrode was analyzed at single-cell level. The luminescence intensity was correlated with the amount of active membrane cholesterol, validating our system for single-cell cholesterol analysis. The relative high standard deviation on the luminescence suggested high cellular heterogeneities on hydrogen peroxide efflux and active membrane cholesterol, which exhibited the significance of single-cell analysis. This success in ECL imaging for single-cell analysis opens a new field in the parallel measurement of surface molecules at single cells.

A BODIPY-derived fluorescent probe for cellular pH measurements.

In this study, BODIPY-appended calix[4]arene was chosen as a fluorescent probe for intracellular pH. The compound with cell permeability can monitor the minor pH change near neutrality inside the cell and is the first BODIPY-derived probe reported for cytosolic pH. Owing to a high level of cell retention and minor cytotoxicity of the probe, stable fluorescence is provided in the cells for 24h, facilitating the precise observation of intracellular pH. A model of cell apoptosis was designed by exposure of the cells to a low concentration of hydrogen peroxide. An increase in the fluorescence of the cells confirmed that BODIPY-appended calix[4]arene sensed the fluctuation of the cellular pH during early cell apoptosis. The developed fluorescent pH probe will be useful for the study of cell apoptosis.

C3N4 Nanosheet Modified Microwell Array with Enhanced Electrochemiluminescence for Total Analysis of Cholesterol at Single Cells

Here, a g-C3N4 nanosheet modified microwell array providing enhanced electrochemiluminescence (ECL) and better visible sensitivity was prepared to simultaneously analyze total (membrane and intracellular) cholesterol at single cells. The detection limit for ECL visualization of hydrogen peroxide at microwell array was improved to be 500 nM that guaranteed the detection of low concentration cholesterol at single cells in parallel. To achieve single cell cholesterol analysis, the individual cells cultured at the microwell array were exposed to cholesterol oxidase generating hydrogen peroxide for luminescence analysis of membrane cholesterol, and then treated with triton X-100, cholesterol esterase, and cholesterol oxidase to produce hydrogen peroxide from intracellular cholesterol for luminescence determination. The observation of the luminescence spots at microwells in these two steps confirmed the codetection of membrane and intracellular cholesterol at single cells. The inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) resulted in less intracellular cholesterol storage (less luminescence) and more membrane cholesterol (more luminescence). The correlation of the luminescence intensity with the amount of cholesterol confirmed that our assay could simultaneously monitor membrane and intracellular cholesterol pools at different cellular states, which should offer more information for the study of cholesterol-related pathways at single cells.

Fast serial analysis of active cholesterol at the plasma membrane in single cells.

Previously, our group has utilized the luminol electrochemiluminescence to analyze the active cholesterol at the plasma membrane in single cells by the exposure of one cell to a photomultiplier tube (PMT) through a pinhole. In this paper, fast analysis of active cholesterol at the plasma membrane in single cells was achieved by a multimicroelectrode array without the pinhole. Single cells were directly located on the microelectrodes using cell-sized microwell traps. A cycle of voltage was applied on the microelectrodes sequentially to induce a peak of luminescence from each microelectrode for the serial measurement of active membrane cholesterol. A minimal time of 1.60 s was determined for the analysis of one cell. The simulation and the experimental data exhibited a semisteady-state distribution of hydrogen peroxide on the microelectrode after the reaction of cholesterol oxidase with the membrane cholesterol, which supported the relative accuracy of the serial analysis. An eight-microelectrode array was demonstrated to analyze eight single cells in 22 s serially, including the channel switching time. The results from 64 single cells either activated by low ion strength buffer or the inhibition of intracellular acyl-coA/cholesterol acyltransferase (ACAT) revealed that most of the cells analyzed had the similar active membrane cholesterol, while few cells had more active cholesterol resulting in the cellular heterogeneity. The fast single-cell analysis platform developed will be potentially useful for the analysis of more molecules in single cells using proper oxidases.

Polymer Dots for Photoelectrochemical Bioanalysis

Different from the most extensively used inorganic quantum dots (Qdots) for the current state-of-the-art photoelectrochemical (PEC) bioanalysis, this work reports the first demonstration of polymer dots (Pdots) for novel PEC bioanalysis. The semiconducting Pdots were prepared via the reprecipitation method and then immobilized onto the transparent indium tin oxide glass electrode for PEC biodetection of the model molecule l-cysteine. The experimental results revealed that the as-fabricated Pdots exhibited excellent and interesting PEC activity and good analytical performance of rapid response, high stability, wide linear range, and excellent selectivity. In particular, the PEC sensor could easily discriminate l-cysteine from reduced l-glutathione (l-GSH). This work manifested the great promise of Pdots in the field of PEC bioanalysis, and it is believed that our work could inspire the development of numerous functional Pdots with unique properties for innovative PEC bioanalytical purposes in the future.

Attomole Antigen Detection Using Self-Electrochemiluminous Graphene Oxide-Capped Au@L012 Nanocomposite

In this work, a self-electrochemiluminous graphene oxide-capped Au@L012 nanocomposite was prepared as the label at carcinoembryonic (CEA) antibody to detect attomole CEA antigen. To maximize the luminescence intensity, L012 molecules (luminol analogue) were linked with poly(diallyldimethylammonium chloride) (PDDA) to form positive charged PDDA&L012 pairs, which were modified on negative charged Au@nafion nanoparticles to construct a Au@nafion@PDDA&L012 (Au@L012) complex. Graphene oxide with carboxyl groups was capped at Au@L012 complex through electrostatic interaction to serve as an effective matrix for the covalent attachment of CEA antibody. As compared with the traditional used Au nanoparticles modified with luminol, ∼740-fold increase of self-luminescence was observed from this new complex so that CEA antigen as low as 0.5 amol at electrode surface was measurable in the absence of any coreactant. Moreover, the nanocomposite was attached with CEA antigen at MCF-7 cells allowing the detection of CEA antigen from 72 cells. The success in the detection of surface antigen at small population of cells suggested the self-electrochemiluminescence nanocomposite as the new and biosafe label for the ECL immunoassay, which might push the application of ECL for the cellular immunoanalysis.

Cholesterol Oxidase/Triton X-100 Parked Microelectrodes for the Detection of Cholesterol in Plasma Membrane at Single Cells

The classic electrochemical analysis of plasma membrane cholesterol at single cells utilizes a cholesterol oxidase modified microelectrode that oxidizes local cholesterol efflux from the plasma membrane to generate hydrogen peroxide for the electrochemical quantification. In this letter, a mixture of cholesterol oxidase and Triton X-100 was filled in the microcapillary that could park at the Pt layer coated tip due to slow hydrodynamic flow. During the contact of the tip with the cellular membrane, Triton X-100 at the tip permeabilized the contacted membrane to release cholesterol for the reaction with cholesterol oxidase. As compared with the linkage of cholesterol oxidase at the electrode surface, the oxidase parked in aqueous solution at the tip had a higher turnover rate resulting in larger electrochemical signal for single cell analysis. More charge collected at acyl-coA:cholesterol acyltransferase (ACAT) inhibited cells supported that this novel detection strategy could monitor the flunctation of membrane cholesterol at single cells. The successful detection of plasma membrane cholesterol at single cells using the oxidase parked microelectrode will provide a special strategy for the fabrication of biosensor that permits the integration of more molecules without functional groups at the electrode to measure active and inactive molecules in the plasma membrane. Moreover, the larger electrochemical signals collected could further increase the spatial resolution for single cell electrochemical analysis.