Recent developments in single-cell analysis

Analytica Chimica Acta 510 (2004) 127–138

Review

Recent developments in single-cell analysis Xin Lu, Wei-Hua Huang, Zong-Li Wang, Jie-Ke Cheng∗ Department of Chemistry, Wuhan University, Wuhan 430072, China Received 21 July 2003; received in revised form 29 December 2003; accepted 16 January 2004

Abstract Cells play a significant role in life activities such as metabolism and signal transduction and so on. The development of cellular research puts forward a severe challenge to analyst, and the study of single-cell has already become the focus of the frontiers in analytical chemistry. Combining capillary electrophoresis (CE) with detection methods such as laser-induced fluorescence (LIF) and electrochemical detection (ED), and mass spectrometry (MS) have particular description for single-cell analysis. Due to extensive research on objects and complex characteristic in the single-cell, scientists have developed more methods and technologies for application in single-cell analysis. Here we reviewed the current approach of technologies of single-cell analysis in manipulation, injection and lysis of single-cell, capillary electrophoresis, imaging analysis, temporal and spatial monitoring on dynamics of single-cells, and the application. © 2004 Elsevier B.V. All rights reserved. Keywords: Single-cell analysis; Capillary electrophoresis; Imaging analysis; Temporal and spatial monitoring

1. Introduction Cells play a significant role in life activities such as metabolism, signal transduction and so on. Highly efficient and sensitive detection of the components in single-cell will explain some important physiological processes. It will also throw light on study and development in the field of biochemistry, cell biology, neurobiology, medicine, pathology, clinic, etc. [1]. Due to the ultra small size of single-cell (diameter 7–200 ␮m, volume fL–nL), ultratrace amount of component (zmol–fmol), ultrarapid biochemical reaction (ms), high sensitivity, high selectivity, high temporal resolution and ultrasmall sampling-volume are necessary for single-cell analysis. In the recent years, single-cell analysis has gone deep into subcellular (local of cytoplast, cellular membrane, vesicular) and single molecule level. The development of single-cell analysis has already become the focus of the frontiers in analytical chemistry [2]. Combining capillary electrophoresis (CE) with detection such as laser-induced fluorescence (LIF) electrochemical detection (ED), and mass spectrometry (MS) have particular

∗ Corresponding author. Tel.: +86-27-87682291; fax: +86-27-87647617. E-mail address: [email protected] (J.-K. Cheng).

0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.01.014

description for single-cell analysis [3]. Due to extensive research objects and complex characteristics of the single-cell, scientists have developed more methods and technologies applied in single-cell analysis. Here we reviewed the current approaches of single-cell analysis in manipulation, injection and lysis of single-cell, capillary electrophoresis, imaging analysis, temporal and spatial monitoring on dynamic of single-cell, and the application. A prospect to the future trend in this field is proposed.

2. Manipulation, injection and lysis of single-cells 2.1. Manipulation Manipulation of transporting and docking is the first step in ordinary assay of single-cell. The techniques currently utilized for the manipulation of single-cell include micropipets, optical traps, and dielectrophoresis. Optical traps can not directly touch cells but can precisely dock and transport single-cell in spatial. Optical trapping was successfully made biomimetic liposomes close up [4]. Strömberg et al. [5] has transport individual cell in the same way through the microchannels into the fusion container, then the cells were fused together with another individual cell in hypo-osmolar fusion medium, but optical trapping rarely provides the facile trapping and electric field often lead to cell damage.

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Fig. 1. Nanoengineered holding pipets: (A) a representation of the pressure controlled; (B) alignment (top) and LIF response (bottom) of an electroporated cell (left) and compared of the fluorescence responses for electroporated (EP) and non-electroporated cells (right).

Micropipets can directly touch cells and the docking strength can be finely controlled by micro operating system, facilitate the manipulation of docking and transporting. The advantage of this method is that manipulation with spatial resolution is simple. Using micropipets by pressure controlled, Rubakhin et al. [6] firstly demonstrated the docking and transporting of single vesicles by adhesion strength difference in vesicle and target surface. But it has poor reproducibility and complex procedures. Wilson et al. [7] have developed a new micropipet with a concave seating surface at the tip. By using optical trapping single-cell was immobilized in the concave seating and then manipulation of docking and transporting was performed by pressure control (Fig. 1). This system shows simple manipulation and better reproducibility. By using simple laboratory syringes and tubes, Yoshida et al. [8] introduced a novel hydrodynamic cell micromanipulation method that can accurately and precisely control individual suspended cells to arrive at the desired sites under microscope through optimization of fluid speed. Recently, microfluidic chips display unusual advantage in transporting and docking of single-cell with the development of chip technology [9,10]. In particular, the dimensions

(10–100 ␮m) of microfludics have made manipulation of the single-cell application popular. Wheeler et al. [11] have developed a single-cell analysis device (Fig. 2). The microfluidic device that integrates valves and pumps on a microchip not only allows the capture of individual cell from the bulk cells suspension but also permits the subsequent perfusion of nanoliter volumes reagents from perfusion chamber onto the selected cells. This technique demonstrates great potential in manipulating and analyzing single-cell on microfluidic chips. Roper et al. [12] have developed a competitive immunoassay for insulin in a microfluidic device with online mixing of reagents, and detected changes insulin secretion from single islets of Langerhans incubated on the chip. We have designed compact channels and microchamber on the microchip, and firstly transported single-cell on a microfluidic chip and docked individual cell in a microchamber and then real-time monitored quanta release of dopamine from the docking cell, designing and manipulation of which is simpler [13]. In addition, trials of using chips in the culture and reaction of the single-cell have also been carried on [14,15]. We expect microfluidic chips to play a more important role in the manipulation of single-cell.

Fig. 2. Single-cell analysis device. (A) Schematic of device: fluidic channels are dark, control channels are light, R1–R5 are reactant inlets, SB and FB are shield and focusing buffer inlets, respectively. Valves are actuated by applying pressure to control inlets V1–V8. Pumps are activated by actuating P1–P3 or P4–P6 in series. (B) CCD image of an individual Jurkat T cell trapped in cell dock. Note the drain channels (arrows) on the dock.

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2.2. Injection Injection of single-cells is the procedure in which single-cells enter the channel of capillary or chip. Injection of whole cell has an abroad application, because it assures 100% cellular components to be injected, and obtains the best detection limit for low content component in cells and can be manipulated facilely, but invert microscope and precise micromanipulator are necessary in manual injection, and injection’s throughput is also poor. In recent years, automatic and continuous injections of single-cells that have a high injection throughput make the analysis of single-cells convenient and establish the base of automated analysis of single-cells. Krylov et al. [16] have designed an automatic cell injector that can inject a whole cell into the capillary. The tip of sampling introduction vertically touches cell suspension and the cell was injected into the capillary by means of pressure- or electrokinetic-drive. The injection time is controlled by an electronic chronometer. Chen and Lillard [17] have developed a high-throughput continued automatic single-cell injection device, as shown in Fig. 3. Sampling capillaries A and electrophoresis separating capillary B are aligned and immersed in the running buffer, cells are continuously pumped into the capillary A by electroosmotic flow following the application of high voltage to the two ends of the capillaries, the lysis of cells is carried out at the junction of capillaries and then the cellular components are injected into capillary B and separated by CE. This method can perform continuous analysis of

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single-cells without the microscope and micromanipulator. But the time interval between injections of two cells could not be precisely controlled, which may induce the overlap of the detected cellular components. In addition, cell chip can analyze multi-single-cells and improve the efficiency of analysis. McClain et al. [18] have demonstrated flow cytometry of the bacterium Escherichia coli on a microfabricated fluidic device. The channels were coated with poly(dimethylacrylamide) to prevent cell adhesion, and the cells were transported electrophoretically by applying potentials to the fluid reservoirs. The cells were electrophoretically focused at the cross of channel, and then were detected by coincident light scattering and fluorescence. Voldman et al. [19] have developed a new microfabricated dynamic array cytometer (␮DAC) (Fig. 4) that allows researchers to sort and hold individual cell for analysis over longer periods of time. Changing operation conditions can make single-cell aggregate in ion traps and get continuous dynamic analysis of single-cell. This device overcomes the disadvantages that a common device is merely analysis to single-cell or multi-cells, and the dynamic process of single-cell or multi-cells were observed at the same time. All these provide a new, rapid and convenient method for the analysis of single-cells. 2.3. Lysis In order to separate and detect the cellular components, lysis is a necessary procedure. Lysis is adopting definite

Fig. 3. Schematic of instrumental set-up for high-throughput CE analysis of single-cells.

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Fig. 4. Cytometer overview: schematic representation of the ␮DAC, showing single-cells loaded onto the cell array chip and one cell, in row 55, column 46, being sorted by the control system into the fraction collectors after acquisition of dynamic luminescence information from the entire array.

methods to lyse cell and release cellular component. Common methods including detergent, hypoosmotic solution, laser pulse and electrical pulse have been induced to disrupt cellular membrane. Methods of detergent and hypoosmotic solution have manifest a simple operation and a high efficiency in disrupting cell membranes in several seconds, and are generally appropriate for the investigation of the statically or slowly changeable cellular components, such as organic molecule, DNA, RNA, or protein. In our study of single-cell, we found the buffer of 0.1% sodium dodecyl sulfate (SDS) detergent greatly influences process of lysis, even the single-cell which has been introduced into capillary would reverse the orientation of electroosmosis fluid and flow out capillary when 0.1% SDS is dispensed in water [20]. We all know many biochemical reactions in single-cell occur in the seconds or even less time scales, such as enzyme. For accurate measurement, complete cell lysis with termination of biochemical reaction in subsecond time periods was required. Allbritton and co-workers [21–23] have developed the laser micropipet system that can terminate cellular reactions on very rapid time scales (33 ms), prevent change of kinase activity in the lysis time, and ensure efficient loading to the cellular content into a capillary. Because of high cost, difficult experiments and safety risks the widespread use of this technique is limited. Recently they have developed a single electrical pulse based on the technique of electroporation to rapidly lyse an adherent cell. When the applied transmembrane voltage exceeds a critical value, cell is disrupted and lysis occurs, therefore rational controlled pulse time and pulse strength are required. This technique, which does not require costly optical components and the time of lysis equals to that of laser pulse technique, may lead to widespread application.

3. Capillary electrophoresis CE is a low volume sampling, and high efficiency separation technique. It is possible to analyze the contents of the components in single-cell, signal transduction mechanism and the differences between cells. CE has already been successfully applied in single-cell analysis. With more CE modes available, as well as the development of technology and theoretic, a number of reviews describing the analysis of single-cells using CE were have been reported [24–27], we only give an outline of the new progresses of CE. Dovichi and co-workers [28–30] have a long-term goal to perform single-cell protein analysis using CE with ultrasensitive sheath-flow LIF detection. They found that cell adhesion leads to low efficiency of cell sampling and poor reproducibility when the slide was coated with hydrophilic polymers such as poly(2-hydrohyethylmethacrylate) (PHEMA), polyvinyl alcohol (PVA). Using novel capillary sodium dodecyl sulfate–dalton electrophoresis they separated and detected more than thirty proteins in individual cell according to the size of proteins. In addition, they have developed a two-dimensional capillary electrophoresis (2D-CE) mode and applied to single-cell separation and detection (Fig. 5). Because single-cell contains many kinds of proteins, they designed different separation modes for the former and latter capillary, making unavailable separated proteins in the first capillary to gain effective separation in the second capillary by means of changing separation mode. In comparing one-dimensional CE with 2D-CE, 2D-CE expands the kinds and scopes of separation, and also improves the efficiency of separation in single-cell analysis. Phillips [31] has studied secretion of cytokines from neuropeptide-stimulated individual lymphocytes, using microdialysis sampling coupled with immunoaffinity CE with LIF detection.

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4.1. Light microscopy

Fig. 5. Schematic of automated two-dimensional separations. A sample undergoes a preliminary separation in a chromatographic column. Fractions are successively transferred to a second column for additional separation. By recording the signal of the detector as a raster pattern, a two-dimensional separation is displayed.

Presently, the combination of CE and high sensitive detectors has undergone a great progress. Due to the high selective and sensitive detection techniques demanding of single-cell analysis, detectors such as electrochemical and laser-induced fluorescence are needed. But the components in single-cells are limited to electroactive and native fluorescent molecules, respectively. Although derivative reagents are often used to tag non-fluorescent molecules, incomplete reactions always occur in the process of tag and make quantitative errors. MS detection can detect multi-components simultaneously and supply additional structural informations, therefore it can analyze complicate components in single-cell such as protein, peptide, etc. in detail. [3]. Demands of the rapid detection of components in single-cell have been satisfied. But the sensitivity of MS is not highly enough to detected components of ultratrace amounts in single-cell, always need to increase sample quantity with the help of enrichment. Additionally, sample type, sample introduction and the difficulty of ionization limit the useable range of MS. In addition, comparing other chromatographic techniques electrophoresis separation with high separation speed, low reagent consumption and simple injection operation has fabricated on the microchip more facilely. More and more scientists are greatly interested on studying this tendency, all the development and application of CE chip further display its tremendous potential in single-cell analysis.

4. Imaging analysis for single-cells A variety of microscopes provide powerful tools for scientist to studying cells [32]. We can directly study the change of cellular configuration and characteristics of cells from information by microscope supplied. Compare biologists and medical scientists, chemists especially analyst focus on the identification and quantitative analysis of the cell image besides obtaining clear cellular images.

Various approaches of light microscopy have already been developed for imaging analysis of single-cells. Recently, light microscopy techniques for live cell imaging have been reviewed by Stephéns and Allan [33]. Fluorescence imaging for single-cell analysis has been reported most frequently. Due to advances in microscopy and optical microscopies, many different kinds of functional fluorescence microscope systems have been designed to suit different requirements of imaging analysis for single-cell. A common fluorescence microscope equipped with a charged coupled device (CCD) camera observed native fluorescence imaging of the distribution of neurotransmitter 5-HT in individual living astrocytes. Cells were tagged by multiple fluorescent dyes. Every cell was encoded with a unique dye and dispersed into a microwell (6 ␮m) using a high-density microwell array. An inverted epifluorescence microscope equipped with a mercury lamp/optical imaging fiber-based array/CCD detector was employed to obtain single living cell array imaging [34], which obviously enhances the efficiency of single-cell imaging. Employing the inverted microscopy with high voltage mercury lamp and double gating photon counter, we have assembled the detection system with fluorescence photon counter. The selectivity of fluorescence reagents is relatively flexible, since the wavelength scale of mercury lamp is wide. Detection limit is 7.0 × 10−10 mol/l for fluorescence dye fluorescein isothiocyanate (FITC), which is comparable to that of the detection system with LIF/CCD or Photomultiplier Tube (PMT). Dubertret [35] encapsulated individual fluorescent semiconductor nanocrystals (quantum dots, QD) in phospholipid block–copolymer micelles, then injected into a living cell. Using a common inverted fluorescence microscope, they obtained stable and vivo imaging in long time. Confocal laser-scanning microscopy (CLSM) can get high-resolution fluorescence images of delicate structures in cells or organs owing to an extra optical filter (pinhole) in the light path [36,37]. Byassee et al. [38], using CLSM, detected the single transferrin molecule labeled with teramethylrhodamine, which absorbed in human HeLa cells. The background of fluorescence in cells is higher than that in buffer solution, but this background is continuous and stable and does not significantly interfere with the measurement of single-molecules. Jaiswal et al. [39] using common fluorescence microscopy and CLSM, studied fluorescence features through selective labeling of cell surface proteins with QDs conjugated to antibodies and observed endocytic and uptake of the QDs. Using fluorescent dye tagging and confocal fluorescent imaging techniques, Song and Stevens [40] found that primary neurons promote oligodendrocyte differentiation whereas astrocytes from postnatal hippocampus can actively regulate neurogenesis from adult neural stem cells, and factors from astrocytes increase the rates of neuronal differentiation and proliferation of progenitors by approximately six-fold and two-fold, respectively. This

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study attracts considerable attention and brings hope to the treatment of elderly dementia at the early stage. Multiphoton fluorescence microscopes excite samples by long wavelength low energy photons. Compared to single photon excitation, it can penetrate deeper into samples, reduces photobleaching, and has minimal optical toxicity and allows long-term observation. Notably, this technique provides powerful capabilities for in situ observation of intact living organisms or tissues the research of in biological and medical science [41,42]. Using two-photon microscopy, Bousso et al. [43] performed real-time analysis of major histocompatibility complex (MHC) at the cellular contacts. Using intravital two-photon microscopy, Miller et al. [44] imaged the locomotion and trafficking of native CD4+ T cells in the inguinal lymph modes, and showed T cells flowing rapidly in the microvasculature and captured individual homing events. Wang et al. [45] has synthesized the two-photon fluorophore (C625) and studied its biological application when chemically linked to a chemotherapeutic agent (AN-152) by using two-photon laser-scanning microscopy. Processes of the receptor-mediated entry of AN-152 into the cell cytoplasm and subsequently into the nucleus were visually showed. Recently, Trachtenberg et al. [46] used green fluorescence protein (GFP) to tag live mouse and drill a pore, they had observed the images changes of individual pyramidal neurons in the mouse barrel cortex over periods of weeks (Fig. 6). They found that some spines appear and disappear. Imaged dendritic segments by serial-section electron-microscopy revealed retrospectively that spine sprouting and retraction are associated with synapse formation and elimination. These changes show that reconstruction of responded neuron connected, as well as plasticity of cortical receptive fields is accompanied by increased synapse turnover. Related experts comment that these results would bring far-reaching influence in brain research. Total internal reflection fluorescence microscopy (TIRFM) notably reduces background signals. One of the most exciting recent developments in recent years is the ability to image single molecules in living cells [47]. Sako et al. [48] labeled epidermal growth factor (EGF) with the fluorescent dye Cy3 and real-time observed signal transduction of epidermal growth factor receptor (EGFR) by visualizing single EGF molecules in the plasma membrane of A431 carcinoma cells by using TIRFM. Seisenberger et al. [49] real-time visualized the infection pathway of single viruses in living cells in real-time by using TIRFM. Systems of

TIRFM are now commercially available, but proficient skill and complicated treatment of data were required. Fluorescence lifetime imaging microscopy (FLIM) has been applied to imaging analysis of pH in living cells and kinase activation. Due to the limitation of resolution, this method makes strict demands in technique and requires complicated mathematical analysis of results, and its prospects appear limited [33]. Sauer and co-workers [50] has performed a series of experiments using FLIM, Confocal spectrally resolved fluorescence lifetime imaging microscopy (SFLIM) images in the red spectral wave length range and fluorescence intensity measurements obtained from living 3T3 mouse fibroblast cells have used to quantitatively detect and time-resolvedly identify the single molecules in living cells, and demonstrate that ∼10–30% of the oligonucleotides cannot diffuse freely inside the nucleus (Fig. 7). Furthermore, quantitative molecular information on a single-cell level can be obtained and presented demonstrate the feasibility of imaging and identifying single molecules in living cells embedded in cell culture medium. This novel technique would find wide application for the study of complicated reaction process in living cells. Scanning near-field optical microscope (SNOM), which has high sensitivity, can also detect single-cells. The spatial resolution of common optical microscopy is limited in 250 nm (half wavelength of visible light), but the spatial resolution of SNOM is approximately 15 nm. It is the only technique for directly mapping optical and spectroscopic properties below the diffraction limit of light [51,52]. This technique can confirm precise locations of fluorescence, and analyze the microstructure and biochemical reaction processes by micro region optical information. It is therefore valued for studying dynamic process of biology system. SNOM has been applied to human erythrocytes, chromosomes and E. coli imaging [53,54], but it can only be applied to surface analysis and gain limited success on soft samples. However, it is more difficult to achieve at the same level of investment in time and training than the more routine techniques like atomic force microscopy (AFM). 4.2. Atomic force microscopy and scanning electrochemistry microscopy (SCEM) AFM has high resolution and simple sample preparation. It can image and obtain three-dimensional surface information under atmospheric or liquid conditions that approach the physiological environment, and also can

Fig. 6. Two-photon images of dendrites in a live mouse, arrows show stable (triangle), semistable (rhombus), and transient (five-pointed star) spines.

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Fig. 7. Fluorescence lifetime image (τ image) of a 3T3 fibroblast cell: (A) fluorescence lifetime image (τ image) calculated from the overall photon counts detected at detectors I and II; (B) fluorescence intensity (detectors I and II) measured in the cytoplasm from 3T3 mouse fibroblast cells.

directly manipulate atoms and molecules at nanometer level [55]. Scha1r-Zammaretti et al. [56] coated atomic force microscopy tips with a polylvinyl chloride (PVC) layer containing an ionophore for potassium to form a potassium-selective AFM tip with the tips in vitro on MDCK-F cells. AFM with tips of carbon nanotube have already applied to comparative study on erythrocyte infected by plasmodium. In recent years, AFM have already become a standard technique for single-cell conformation analysis. Electron microscope has high spatial resolution, which can observe ultrastructures of cells and can characterize molecular structures [57]. Conventional transmission electron microscope and scanning electron microscope have been applied to single-cell analysis, but cannot be applied in living cell analysis. The relatively new scanning electrochemical microscope equipped with ultramicro-electrode (UME) probe can measure electrochemical character of single-cell while imaging cells. Thus, single-cell can be monitored temporally and spatially [58]. Zhou et al. [59] first applied SECM to biology and single molecule detec-

tion and has performed a series of studies. SECM is mostly used for monitor activity of single-cell. Torisawa et al. [60] has investigated the change of the extracellular oxygen during photosynthesis and respiratory activity in living cells by SECM, and groped for the respiratory activity. Mirikin and co-workers [61,62] has measured redox activity, acid-base reaction, and topography by SECM. The study of charge transfer mechanism in single human breast cell, and the intracellular redox activity in single-cell by SECM were also reported recently. Liebetrau et al. [63] using the negative feedback mode of a SECM successfully identified six biocompatible redox mediators from a large pool of candidates and imaged living PC12 cells before and after exposure to nerve growth factor (NGF). And their studies demonstrate that carbon fiber electrodes with reduced tip diameters can be used for imaging both the cell proper and these neurites (Fig. 8). When exposed to NGF, cells differentiate into a neuron phenotype by growing narrow neurites (1–2 ␮m wide) that can extend >100 ␮m from the cell proper. So changes in the morphology of undifferentiated PC12 cells

Fig. 8. Optical and SECM images of differentiated PC12 cells. The SECM images were recorded at the boxed areas indicated in the optical micrograph using a polymer-insulated flame-etched CFE in 1.0 mM Ru(NH3 )6 3+ .

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could be detected in real-time with the SECM. Thus, combination SECM with electrochemical techniques will make for a powerful tool for the investigation the neurotransmitter dynamics, synaptogenesis, and neurodegeneration of single-cells in culture. 4.3. Other microscopes Ewing and co-workers [64,65] have performed a series of researches on single-cell by using time-of-flight secondary ion mass spectrometry (TOF-SI-MS). As an example, the molecular species around the surface of single-cell of Paramecium organism were accurately observed with this method. They comparatively analyzed biology imaging of freeze-fractured cells by using bright field, scanning ion, and fluorescence microscopy. Single-cell was labeled by fluorescence dye DiI and located by fluorescence microscopy. TOF-SI-MS imaging reveals the localization of membrane lipids of single PC12 cells. Moreover, scanning thermal lens microscope [14], cofocal Raman microscope [66,67] and other microscopy techniques were also applied to single-cell analysis. With the development of green fluorescence protein and biological imaging software, imaging analysis of single-cells will obtain more advantages from the fluorescence resonance energy transfer (FRET), multicolor imaging and multi-dimensional imaging [68,69].

5. Real-time dynamic monitoring of single-cells Dynamic processes of single-cell have important influence upon morphology, function, metabolism, proliferation, differentiation and survival of single-cell. Studying the dynamic process of single-cell has very important meaning in several subject fields, such as neurobiology, cell biology, clinic, pathology, pharmacody, etc. By means of the recent development of analysis techniques, we can real-time dynamically monitor the signal molecules release from single living cell, even from individual vesicle, with temporal and spatial resolution. Electrochemical detection with the ultra-microelectrodes is the main method to real-time monitoring release from single-cells [70]. Recently, it mostly focuses on two aspects, the increase in detection sensitivity and the application of microchip techniques. Combining ultra-microelectrodes with a patch clamp, Hochstetler et al. [71] detected the dopamine release from individual neurons of the mouse retina in real-time. Recently, they real-time measured the active transport of electroactive neurotransmitter dopamine into a single-cell HEK-293 in a picoliter vial [72]. Yasukawa et al. [73] has developed a new picoliter-volume electrochemical analytical chamber, which integrated the working microelectrode. The flux of metabolite released from a single-cell was estimated by using electrochemical-linked assays, and the activity of the cellular protection mechanisms was also

Fig. 9. Amperograms of vesicular exocytosis from PC12 cell: (A) cartoons of cell-microelectrode and amperograms of vesicular exocytosis from PC12 cell induced by stimulating the cell with 1 mmol/l nicotine obtaining from a carbon fiber microelectrode; (B) cartoons of cell-nanoelectrode and amperograms of vesicular exocytosis from PC12 cell induced by stimulating the cell with 1 mmol/l nicotine obtaining from a carbon fiber nanoelectrode.

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investigated. Chen et al. [74], using microsystems technology, fabricated electrochemical electrodes in picoliter-sized wells on the chip array. Placing a single-cell into a microwell, they measured the catecholamine released from individual cell with milliseconds resolution. This method greatly simplifies the whole process of single-cell detection. We have developed a new type low noise carbon fiber nanoelectrodes [75], and the nanoelectrodes have been used to real-time monitored dopamine released from single PC12 cells in temporal and spatial resolution (Fig. 9). We firstly detected dopamine release from individual vesicle in single-cell and proved non-uniform distribution of dopamine in single-cell [76]. Optical methods, which can monitor release of nonelectroactive components from cells, are powerful tools in real-time monitoring of the release from single-cell. LIF, which has high sensitivity and selectivity, can be used to quantitatively measure release from cells. Recently, Anderson et al. [77,78] has monitored zeptomole-abundant doxorubicin (DOX) metabolites in single-cell by using sensitive CE-LIF, and compared distribution of these metabolites in three subcellular fractions. These methods can detect the unidentified zeptomole DOX metabolites, define their subcellular localization and provide a more complete description of their metabolic significance. But it is difficult to simultaneously monitor the cells in temporal and spatial. Microscopy technique is especially outstanding in this aspect. Combined GFP labeling and different fluorescence microscopes can study a series of about dynamic characters such as: endocytosis, phagocytosis of single-cell [79,80]. Using laser-scanning confocal microscopy combined with amperometric method, Kennedy and co-workers [81,82] measured Zn2+ efflux from single pancreatic ␤-cell with spatial and temporal resolution and discussed exocytosis mechanism of single pancreatic ␤-cell. Angleson et al. [83] have developed an optical assay for detection secretion from isolated rat pituitary lactotrophs that permits the detection of single exocytic and endocytic events over the entire surface of cell. By using an ordinary epifluorescence, combining optical- and electron-microscopy, they observed the recordings about electrophysiological of membrane capacitance. In addition, by using an enzyme-based self-referencing glucose microsensor, glucose consumption of pancreatic ␤-cells was measured by Jung et al. [84]. The real-time dynamic movements of glucose in a biological microenvironment were first recorded.

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6.1. Proteins Proteins contribute constitute ∼18% of the total weight in mammal cells. They catalyze lots of chemical reactions, support the skeletons of different structures, control the permeability of the membrane, modulate the concentration of metabolize, identify other biomolecules and control genes etc. At present, molecule diagnoses of disease and rapidly arisen studies on proteomics have urgently demanded the development of a brand-new method to solve the problems of numerous different proteins analysis in single-cell. Dovichi and co-workers [28,29,85,86] have a long-term goal to perform proteomics analysis in single-cells. At present, 2D-CE capillary electrophoresis is a kind of competitive new method besides commonly used two-dimensional gel electrophoresis. They have made great efforts and gained one-dimensional electropherograms of more than 30 kinds of protein in single human colon adenocarcinomas of proteins cell HT29, the whole analysis time was only 45 min, and the detection limit reached 10−10 to 10−11 mol/l. Mass detection limit of two-dimensional separation and detection reached zmol level. Recently, they have analyzed the protein expression of a single-cell at a known phase of the cell cycle. This study provides unambiguous information on the cell-to-cell variation in protein expression at each stage in the cell cycle. Proteomics is a dynamic concept, and has important theoretic significance in studying proteinic actions and revealing essential rules. In addition, it is the basis of structure of molecule design and medicinal exploring. Umezawa and co-workers [87–89] designed and synthesized a new environment-sensitive fluorescent probe that was capable of being attached at its genetically engineered 4Cys motif of recombinant proteins inside live cells. By using this fluorescent probe, the conformational changes of proteins were imaged in live cell. Subsequently combing developed genetically encoded fluorescent indicators and FRET technique, they visualized protein phosphorylation by the insulin receptor. Based on the protein splicing-induced complementation, they designed split luciferase as an optical probe to detect protein–protein interactions in intact mammalian cells. Fluorogenic enzyme substrates and receptor ligands were rapidly delivered to cells by electroosmosis and internalized by electroporation, Nolkrantz et al. [90] observed the function of intracellular proteins in single-cell. 6.2. Kinases

6. Application of single-cell analysis In recent years, single-cell analysis has been developed from analysis of cell contents to membrane analysis, individual vesicles in cells, and even single molecule analysis in single-cells, and its application is also continuously increasing. We think the important realm of application in single-cell analysis currently is as follows:

Kinases in cells relate closely to performing various functions by cells. The concentration of kinases in cell is low and would rapidly change in a short time when the environment changes. Kinases easily lose their activity, so detecting all sorts of kinases and their activities in cells must be rapid and available [91]. Allbritton and co-workers [92–94] realized that multiple kinases and their activities could be measured simultaneously in single-cell, which opens a window

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for understanding the molecule mechanisms responsible for cellular control and behavior, and reveal the dynamic nature of signal transduction networks in living cells. Combining the capability of multiparametric flow cytometry and phospho-spectic antibodies of various kinases, Perez and Nolan [95] detected four distinct kinases of the MAPK family in single-cells. Based on phosphorylation-induced changes in FRET and three genetically encoded fluorescent reporters for the tyrosine kinases Src, Abl, and epidermal growth factor receptor, Ting et al. [96] non-destructively imaged dynamic protein tyrosine kinase activities with high spatial and temporal resolution in single living cell. Combination of the on-capillary enzyme-catalyzed reaction and electrochemical detection at a carbon fiber disk bundle electrode, Sun and Jin [97] have analyzed enzymes at the zeptomole level in single-cell recently. 6.3. Peptides Peptides are polypeptides active substances that can be synthesized, stored and released by nervous system. They are important chemical signal messengers in multi-cell organism. Studying the rules of the changes in neurotransmitter and neuromodulator is important to fully understand the learning and memory mechanisms of central nerve system. MS detection provides structural information and molecular weight of peptides in single-cells simultaneously [98]. To solve the problems of neuropeptide detection in neurosciences, Sweedler’s group [99–101] has processed systematic and explored works about neuropeptide analysis in single-cells. To combine matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-MS) with different techniques of sample’s preparation and separation, etc. such as immunochemistry, capillary electrophoresis, they successfully detected neuropeptides in single neurons, even in individual vesicles, and obtained integrated separate skeleton diagram of neuropeptides. In addition, they identified the peptides released from single neurons, which formed from the common neuronal model Aplysia californica. 6.4. Single molecule detection Direct observation of single molecule and single molecular event in the living cells could dramatically improve our understanding to the basic cellular processes as well as improving our knowledge of the intracellular transport and the fate of therapeutic agents. Dynamically monitoring single molecule in single living cell has huge challenges to analysis chemistry. Using single molecule imaging techniques, Sako et al. [48] detected the location, movement, interaction and biochemical reaction of single EGFR molecules and observed the presence of a preformed EGFR dimer and conformational fluctuation of this dimer. This dimerization of the EGFR is known to be essential to signal transduction. Byassee et al. [38] detected the single transferrin molecule

labeled with teramethylrhodamine, which absorbed by human HeLa cells. Recently, it is reported that Knemeyer at al. [50] have detected and identified single mRNA molecules in living mouse fibroblast cells 3T3. This research provides novel approaches for investigating chemical reaction and observing changes in configuration in the living cells. 6.5. Nucleic acid Nucleic acid analysis in single-cell is greatly significant not only in molecular biology but also in diagnosis of disease at early stage. Yeung and co-workers [102,103] has developed reverse transcription-polymerase chain reaction (RT-PCR)/CE/LIF integrated system that can directly detect genes from single-cell. All of the operations, including lysis of cells, deoxyribonuclease (DNase) treatment, RT-PCR, CE/LIF detection, etc., were completed in this system within 3 h. This system is available to selectively analyze genes of single-cells. Lillard and co-workers [104,105] detected low molecular mass RNAs in individual Chinese hamster ovary cell by using CE/LIF. After this, the expression of ␤-actin in individual mammalian cell was monitored by using CE/LIF and the reverse transcriptase-polymerase chain reaction. Perlette and Tan [106] has developed molecular beacon (MB), an oligonucleotide probe with inherent signal transduction mechanisms. And by using molecular beacons and fluorescence imaging system, the real-time and ultrasensitive detection of mRNA hybridization and the visualization of oligonucleotide/mRNA interactions inside single living kangaroo rat kidney cells were achieved. 6.6. Diagnosis and therapy of diseases Single-cell analysis gives great assistance to diagnosis and therapy of diseases, the state of diseases and effects of treatments can be made certain through analyzing the change of components and contents, even DNA fragments in single-cell. Due to large variations of the isoenzyme ratios among individual cells, Xue and Yeung [107] measured the lactate dehydrogenase (LDH) isoenzyme activities and the relative ratios between different LDH isoenzymes by using CE/LIF; they expected that this method could be applied to early diagnosis of leukemia. Vo-Dinh et al. [108] measured benziopyrene tetrol (BPT), a metabolite of the carcinogen benzo[a]pyrene (BaP) and of the BaP–DNA adduct, with the help of an antibody-based nanoprobe. BPT can serve as a biomarker for monitoring DNA damages due to BaP exposure and possible precancer diagnosis.

7. Future directions Without question, single-cell analysis is one of the focus fields of analytical chemistry in this century. Single-cell analysis is not only the frontier of academic development, but also has close relation to human health. Thus, it has

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important academic significance and tremendous applied potential. Although people have made a series of works, lots of problems need to be investigated and discussed. Single-cell analysis is closely related to many subjects, such as chemistry, life science, clinic medicine, physics, micro-photology, microelectronics, computer science, material science, etc. To integrate advances of different subjects, and perform real-time, online, in situ, in vivo, highly resolved, highly selective studies on single-cell or the subcellular level. The complete automation of analysis process may be achieved. With the help of advanced techniques such as: physics, electronics, micro-process, etc., single-cell analysis may be manipulated simply and fast to perform the study on single-cells. Bio-imaging techniques applied to single-cell analysis will achieve precise, high-throughput, automatic analysis of dynamic changes in intracellular component location and content in single-cells. On the other hand, due to the successful research of QDs, and clear observation of the slow dynamic process in long time become probable. Thanks to the development of nanoprobe, we can analyze the interesting location inside single-cell by inserting a probe into a single-cell. It is an extremely developed tool for in situ single-cell analysis. To fulfill the project of human genome ahead, proteomics analysis has become a focus study in science realm. Studies on genome, proteomics, even carbohydrates in single-cell level, are not only a significant research also a challenge project. Investigation of intracellular components and reaction kinetics of single-cell provides a powerful help to researches and applications in biology, medicine, pathology, clinic, etc. Chip assay has already been widely applied to dynamic analysis of genes and proteins. The development of micro total analysis system (␮-TAS) in recent years provides excellent manipulation and detected platform for single-cell analysis; it makes fast complete manipulation and analysis of single-cell available. As a consequence, the micromation and automation of single-cell analysis may be realized. Single-cell analysis is a very important research field, and is the frontier of science. With continuous progress of single-cell analysis, we believe it will promote the development of the whole life science.

Acknowledgements We gratefully acknowledge the support of the China National Natural Science Foundation (Grant No. 20299034).

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