•Multiplexed optical DNA coding nanobeads (MOCNBs) are fabricated.•The MOCNBs can serve as labels for single-molecule counting analysis (SMCA).•The limit of detection of the SMCA using MOCNBs is 1.3 × 10−15 mol L−1.•The SMCA using MOCNBs can be used in multi-gene expression analysis.A method for fabrication of multiplexed optical coding nanobeads (MOCNBs) was developed by hybridizing three types of coding DNAs labeled with different dyes (Cy5, FAM and AMCA) at precisely controlled ratios with biotinylated reporter DNA modified to magnetic streptavidin-coated nanobeads with a diameter of 300 nm. The color of the MOCNBs could be observed by overlapping three single-primary-color fluorescence images of the MOCNBs corresponding to emission of Cy5 (red), FAM (green) and AMCA (blue). The MOCNBs could be easily identified under a conventional fluorescence microscope. The MOCNBs with different colors could serve as the multiplexed optical coding labels for single-molecule counting analysis (SMCA) and be used in multi-gene expression analysis (MGEA). In the SMCA-based MGEA technique, multiple messenger RNAs (mRNAs) in cells could be simultaneously quantified through their complementary DNAs (cDNAs) by counting the bright dots with the same color corresponding to the single cDNA molecules labeled with the MOCNBs. We measured expression profiles of three genes from Lepidoptera insect Helicoverpa armigera in ∼100 HaEpi cells with and without steroid hormone inductions to demonstrate the SMCA-based MGEA technique using MOCNBs.

•A single-molecule-detection (SMD) microarray for 10 samples is fabricated.•The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample.•The limit of detection of SMA is as low as 1.3 × 10−16 mol L−1.•The SMA can be applied in single-cell multiple gene expression analysis.We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3 × 10−16 mol L−1. The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three types of cDNAs corresponding to beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein, large, P2 mRNAs in single human breast cancer cells and 5 random synthetic DNAts are simultaneously quantified to examine the SMA and SMA-based single-cell multiple gene expression analysis.

In this work, electrochemiluminescence resonance energy transfer (ECRET) between CdSe/ZnS quantum dot (QD) as the donor and cyanine dye (Cy5) as the acceptor in the conjugates consisting of QD, DNA and Cy5 was studied in detail. When a negative potential was applied to the conjugates immobilized on an Au electrode, QDs emitted a light with a maximum emission (λm) of 590 nm or transfered energy to proximal ground-state Cy5 molecules in 0.1 mol/L phosphate buffer (pH 7.4) containing 0.1 mol/L K2S2O8 and 0.1 mol/L KNO3. The excited state Cy5 molecules relaxed to their ground state by emitting a light with a λm of 675 nm. ECRET between QD and Cy5 in the conjugates could be used to evaluate the interactions between DNAs and to measure the conformational changes of DNAs as well as the distances between groups in DNAs. For the ECRET system, ECRET efficiency was high due to high Cy5-to-QD ratio and low Förster distance.Graphical abstractElectrochemiluminescence resonance energy transfer in the conjugates consisting of CdSe/ZnS QD, DNA and Cy5 dye.Highlights► Electrochemiluminescence resonance energy transfer (ECRET) between QD and Cy5. ► Evaluation of the interactions between DNAs using ECRET between QD and Cy5. ► Measurement of the conformational changes of DNAs using the ECRET system. ► Measurement of distances between groups in DNAs using the ECRET system.

A new signal amplification strategy based on DNA hybridization–dehybridization reaction on the surface of magnetic submicrobeads (MSBs) for fluorescence detection of ultrasensitive DNA was developed. In this strategy, MSBs modified with probe DNA (DNAp-MSBs) were bound to target DNA (t-DNA) (with a ratio of 1:1) captured to a substrate. The DNAp-MSBs were released from the substrate via DNA dehybridization and then hybridized with Cy5-labeled detection DNA (Cy5-DNAd). After the Cy5-DNAd and DNAp-MSBs were separated by dehybridization, the Cy5-DNAd was collected. The DNAp-MSBs were then hybridized with other Cy5-DNAd to initiate the next hybridization–dehybridization round. This recycling of the hybridization–dehybridization process on the surface of the DNAp-MSBs was repeated multiple times to accumulate Cy5-DNAd. Finally, fluorescence intensity of the collected Cy5-DNAd was measured. Using this strategy, the limit of detection for determination of t-DNA was 8.5 × 10−15 mol L−1 for 11 cycles. The ultrasensitive assay was used to quantify ribosomal protein, large, P2 (RPLP2) mRNA in human breast cancer cells.

In this paper, we reported an ultrasensitive ECL spectrometry for determination of DNA using magnetic streptavidin-coated nanobeads MNBs (SA-MNBs) as the carrier of Ru(bpy)32+-NHS, where bpy = 2,2′-bipyridyl and NHS = N-hydroxysuccinimide ester, to amplify signal. The SA-MNBs were conjugated to the hybrids consisting of capture DNA, target DNA (t-DNA) and probe DNA immobilized on a substrate, followed by releasing the SA-MNBs and binding a huge number of Ru(bpy)32+-NHS to the SA-MNBs. The SA-MNBs with Ru(bpy)32+-NHS were immobilized on an Au film electrode by means of a magnet. In the presence of tri-n-propylamine, the ECL spectrum of the Ru(bpy)32+-NHS at 1.35 V was acquired by using an optical multi-channel analyzer. The maximum emission intensity on the ECL spectrum was used to quantify DNA. Using this method, not only the limit of detection for DNA determination was as low as 1.2 × 10−15 mol/L, but also the ECL spectrum of Ru(bpy)32+-NHS on the surface of the SA-MNBs was obtained. The ultrasensitive ECL spectrometry could be used to measure gene expression level in cells.Highlights► A new electrochemiluminescence (ECL) technique, ECL spectrometry, is reported. ► A new method for determination of 1.2 × 10−15 mol/L DNA is developed. ► The ECL spectrometry is used to measure gene expression level in cells.

Novel electrochemiluminescence resonance energy transfer (ECRET) between an emitter electrochemically generated by luminol as the donor and luminescent quantum dots as the acceptor is investigated. The ECRET technique can be used to study the interactions and conformational changes of proteins.

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 × 10−14 mol L−1. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

Electrochemiluminescence resonance energy transfer (ECRET) between CdSe/Zns quantum dots (QDs) as the donor and cyanine dye (Cy5) molecules as the acceptor in QD-Cy5 conjugates with DNA or protein as the linker was reported. When a negative potential was applied, the excited-state CdSe/ZnS* was produced in 0.1 mol/L phosphate buffer (pH 7.4) containing 0.1 mol/L K2S2O8 and 0.1 mol/L KNO3 (PB-K2S2O8). The CdSe/ZnS* went back to the ground-state CdSe/ZnS to emit light at 590 nm or to transfer energy to proximal ground-state Cy5 molecules. The resultant excited-state Cy5 molecules relaxed to their ground state by emitting a light at 675 nm. The ECRET between QDs and Cy5 was used to evaluate interactions between DNAs and to measure conformational changes of DNAs and proteins.Electrochemiluminescence resonance energy transfer between quantum dots as the donor and Cy5 dye molecules as the acceptor in QD-Cy5 conjugates with DNA or protein as the linker.Highlights► Electrochemiluminescence (ECL) spectra of CdSe/ZnS quantum dots are measured. ► ECL resonance energy transfer (ECRET) between quantum dot and Cy5 dye is studied. ► The ECRET system is used to evaluate interactions between DNAs. ► The ECRET system is used to measure conformational changes of DNA or protein. ► The ECRET system is used to measure distances between groups in biomolecules.

We developed an ultrasensitive electrochemiluminescence (ECL) method for DNA determination using magnetic submicrobeads (SMBs) as the carrier of Ru(bpy)32+ (bpy = 2,2′-bipyridy) and carbon nanotubes (CNTs) as accessorial electrode material. The SMBs with Ru(bpy)32+ were wrapped with CNTs and then immobilized on an Au electrode. In the presence of tri-n-propylamine, ECL of the Ru(bpy)32+ on the SMBs was detected. Since one target DNA (t-DNA) molecule corresponded to one SMB with a large number of Ru(bpy)32+, the ECL signal was amplified. In addition, the Ru(bpy)32+-loaded SMBs were wrapped with CNTs that contacted the electrode. The ECL of Ru(bpy)32+ was greatly increased. Using this method, t-DNA of 3 × 10−16 mol/L could be detected. The method could be used to quantify mRNA in cells.Highlights► Electrochemiluminescence (ECL) of Ru(bpy)32+-coated submicrobeads wrapped with carbon nanotubes is measured. ► Ultrasensitive ECL method for determination of 3 × 10−16 mol/L DNA is investigated. ► The ECL method is used to quantify mRNA in cells.

An ultra-sensitive assay for quantification of DNA based on single-molecule detection coupled with hybridization accumulation was developed. In this assay, target DNA (tDNA) in solution was accumulated on a silanized substrate blocked with ethanolamine and bovine serum albumin (BSA) through a hybridization reaction between tDNA and capture DNA immobilized on the substrate. The tDNA on the substrate was labeled with quantum dots which had been modified with detection DNA and blocked with BSA. The fluorescence image of single QD-labeled tDNA molecules on the substrate was acquired using total internal reflection fluorescence microscopy. The tDNA was quantified by counting the bright dots on the image from the QDs. The limit of detection of the DNA assay was as low as 6.4 × 10−18 mol L−1. Due to the ultra-high sensitivity, the DNA assay was applied to measure the beta-2-microglobulin messenger RNA level in single human breast cancer cells without a need for PCR amplification.

An ultra-sensitive DNA microspot assay was developed that required 1.8 nL samples and was based on single-molecule detection. The solution of the target DNA (tDNA) was spotted onto the coverslip modified with capture DNA (DNA1) and blocked with ethanolamine and bovine serum albumin using a pintool type microspoting robot. The microspot had a diameter of ~300 μm. The tDNA was captured by the DNA1, and the tDNA was then labeled with a detection DNA that previously was labeled with a quantum dot. Next, a fluorescence microscopic image of the microspot was acquired using a single-molecule microspot reader during total internal reflection fluorescence excitation. As little as 4 × 10−22 mole (240 molecules) of tDNA can be detected by this method. The response is linear in the range from 6.0 × 10−22 to 1.2 × 10−19 mole of tDNA. All operations (including the acquisition of microspot images and single-molecule counting) were performed using the MetaMorph software. The assay was applied to the determination of osteopontin messenger RNA in single decidual stromal cells without the need for PCR amplification.

An ultrasensitive solid-phase fluorescence resonance energy quenching (FREQ) method for determination of 1,4-dihydroxybenzene (DHB) using mercaptosuccinic acid (MSA)-capped CdTe quantum dots (QDs) immobilized on silica nanoparticles (NPs) as donors was developed. In the method, silica NPs were first modified with 3-aminopropyltriethoxysilane (APTS). Then, MSA-capped CdTe QDs were immobilized on the surface of the APTS-modified silica NPs. Finally, DHB in the solution was attached to the empty sites on the surface of silica NPs with QDs through electrostatic interaction. The fluorescence emission of the QDs was quenched by the proximal DHB molecules on the silica NPs. The quenching efficiency of the solid-phase FREQ method was 200-times higher than that of the solution-phase FREQ method. Using the ultrasensitive solid-phase FREQ method, DHB as low as 2.4 × 10−12 mol/L could be detected. The method was applied to quantify trace DHB in water samples.

A novel ultra-sensitive single-molecule-counting microarray assay (SMCMA) with a 1.8-nL sample volume for quantification of proteins was provided using total internal reflection fluorescence microscopy coupled with quantum dot (QD)-labeling. In the SMCMA, the microarray consisting of ∼300 μm diameter microspots with the spot-to-spot pitch distance of 500 μm was fabricated by spotting 1.8 nL of solutions containing the target protein onto the substrate which was modified with primary antibody of the protein and blocked with ethanolamine and BSA using a pin-tool type microarraying robot. Then, biotinylated secondary antibody of the protein was bound to the protein to form sandwich immunocomplexes. After labeling with streptavidin-coated QDs, the whole image of the microarray was acquired using a homemade single-molecule microarray reader. The target protein was quantified based on the number of bright dots from the QDs corresponding to single target protein molecules on the microarray. Using the SMCMA, an amount as low as 1.5 × 10−21 mole (904 molecules) for proteins could be detected. The SMCMA was applied to measure dynamic expression of osteopontin in living cells.

A new electrochemiluminescence (ECL) DNA assay is developed using quantum dots (QDs) as DNA labels. When nanoporous gold leaf (NPGL) electrodes are used, sensitivity of the ECL assay is remarkably increased due to ultra-thin nanopores. In this assay, target DNA (t-DNA) is hybridized with capture DNA (c-DNA) bound on the NPGL electrode, which is fabricated by conjugating amino-modified c-DNA to thioglycolic acid (TGA) modified at the activated NPGL electrode. Following that, amino-modified probe DNA is hybridized with the t-DNA, yielding sandwich hybrids on the NPGL electrode. Then, mercaptopropionic acid-capped CdTe QDs are labeled to the amino group end of the sandwich hybrids. Finally, in the presence of S2O82− as coreactant, ECL emission of the QD-labeled DNA hybrids on the NPGL electrode is measured by scanning the potential from 0 to −2 V to record the curve of ECL intensity versus potential. The maximum ECL intensity (Im,ECL) on the curve is proportional to t-DNA concentration with a linear range of 5 × 10−15 to 1 × 10−11 mol/L. The ECL DNA assay can be used to determine DNA corresponding to mRNA in cell extracts in this study.

An ultrasensitive electrochemical method for determination of DNA is developed based on counting of single magnetic nanobeads (MNBs) corresponding to single DNA sequences combined with a double amplification (DNA amplification and enzyme amplification). In this method, target DNA (t-DNA) is captured on a streptavidin-coated substrate via biotinylated capture DNA. Then, MNBs functionalized with first-probe DNAs (p1-DNA−MNBs) are conjugated to t-DNA sequences with a ratio of 1:1. Subsequently, the p1-DNA−MNBs are released from the substrate via dehybridization. The released p1-DNA−MNBs are labeled with alkaline phosphatase (AP) using biotinylated second-probe DNAs (p2-DNAs) and streptavidin−AP conjugates. The resultant AP−p2-DNA−p1-DNA−MNBs with enzyme substrate disodium phenyl phosphate (DPP) are continuously introduced through a capillary as the microsampler and microreactor at 40 °C. AP on the AP−p2-DNA−p1-DNA−MNBs converts a huge number of DPP into its product phenol, and phenol zones are produced around each moving AP−p2-DNA−p1-DNA−MNB. The phenol zones are continuously delivered to the capillary outlet and detected by a carbon fiber disk bundle electrode at 1.05 V. An elution curve with peaks is obtained. Each peak is corresponding to a phenol zone relative to single t-DNA sequence. The peaks on the elution curve are counted for quantification. The number of the peaks is proportional to the concentration of t-DNA in a range of 5.0 × 10−16 to 1.0 × 10−13 mol/L.

We developed an ultrasensitive quantitative single-molecule imaging method for fluorescent molecules using a combination of electrochemical adsorption accumulation and total internal reflection fluorescence microscopy (TIRFM). We chose rhodamine 6G (R6G, fluorescence dye) or goat anti-rat IgG(H+L) (IgG(H+L)-488), a protein labeled by Alexa Fluor 488 or DNA labeled by 6-CR6G (DNA-R6G) as the model molecules. The fluorescent molecules were accumulated on a light transparent indium tin oxide (ITO) conductive microscope coverslip using electrochemical adsorption in a stirred solution. Then, images of the fluorescent molecules accumulated on the ITO coverslip sized 40 × 40 µm were acquired using an objective-type TIRFM instrument coupled with a high-sensitivity electron multiplying charge-coupled device. One hundred images of the fluorescent molecules accumulated on the coverslip were taken consecutively, one by one, by moving the coverslip with the aid of a three-dimensional positioner. Finally, we counted the number of fluorescent spots corresponding to single fluorescent molecules on the images. The linear relationships between the number of fluorescent molecules and the concentration were obtained in the range of 5 × 10−15 to 5 × 10−12 mol/L for R6G, 3 × 10−15 to 2 × 10−12 mol/L for IgG(H+L)-488, and 3 × 10−15 to 2 × 10−12 mol/L for DNA-R6G.

Scanning electrochemical microscopy (SECM) is a powerful tool to examine the respiratory activity of living cells. However, in SECM measurements of cell respiratory activity, the signal recorded usually also includes the signal corresponding to the cell topography. Therefore, measurements of cell respiratory activity using conventional SECM techniques are not accurate. In the present work, we develop a method for accurate measurement of the respiratory activity of single living cells using SECM. First, cells are immobilized on a glass substrate modified with collagen. Then, a Pt ultramicroelectrode tip of SECM held at −0.50 V is scanned along the central line across a living cell and a SECM scan curve, i.e., the relationship of the tip current versus the displacement (the first scan curve) is recorded with a negative peak. The peak current ip on this first scan curve is composed of ip1, which corresponds to the cell respiratory activity and ip2, which corresponds to the cell topography. In order to isolate the ip2 component, the cell is killed by exposing it to 1.0 × 10−3 mol/L KCN for 10 min. The tip is then scanned again with the same trace over the dead cell, and a second SECM scan curve is recorded. Noting that the topography of the dead cell is the same as that of the living cell, this second scan curve with a negative peak corresponds now only to the cell topography. Thus, ip2 is obtained from the second SECM scan curve. Finally, ip1 corresponding to the respiratory activity of the living cell can be accurately calculated using ip1 = ip − ip2. This method can be used to monitor real-time change in the respiratory activity of single cells after exposing them to KBr, NaN3 and KCN.

We developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy with adsorption equilibrium. In this method, the adsorption equilibrium of protein was achieved between solution and glass substrate. Then, fluorescence images of protein molecules in a evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally, the number of fluorescent spots corresponding to the protein molecules in the images was counted. Alexa Fluor 488-labeled goat anti-rat IgG(H + L) was chosen as the model protein. The spot number showed an excellent linear relationship with protein concentration. The concentration linear range was 5.4 × 10−11 to 8.1 × 10−10 mol L−1.

This paper uses scanning electrochemical microscopy (SECM) coupled with an intracellular standard addition method to quantify enzyme activity in single intact cells. In this work, peroxidase (PO) inside human neutrophils is chosen as the model system. Cells immobilized onto a silanized coverslip are perforated with digitonin to form micropores on the cell membrane. Hydroquinone (H2Q) and hydrogen peroxide (H2O2) as the enzyme substrates diffuse through the micropores into the cell interior. There, H2Q is converted into benzoquinone (BQ) by intracellularPO. BQ diffuses with a steady flux through the micropores from the cell interior onto the cell surface. The BQ near the cell surface is detected by the Au tip of SECM held at –0.3 V. When the tip is scanned laterally along the central line over the cell, a 2-D scan curve is obtained. Then, the intracellular standard addition method using ultramicroinjection with a submicrometer-sized micropipette tip is performed. After ultramicroinjection of a standard solution, another 2-D scan curve is recorded. The intercellular enzyme activity can be calculated from both peak current on two scan curves. This method to quantify PO activity in the cell environment has several obvious advantages: high sensitivity due to signal amplification viaintracellularenzyme-catalyzed reaction and no sample dilution, no electrode fouling from adsorption of intracellular biological molecules, and no interference from electro-active compounds that can be directly oxidized at the SECM tip or from oxygen in the detected solution.

We developed a new simple approach to fabricate dual-disk electrodes with a nanometer-radius electrode and a micrometer-radius electrode. First, nanometer-sized electrodes and micrometer-sized electrodes were constructed using 10-μm-radius metal wires, respectively. To fabricate the nanometer-sized electrode, after the apex of the 10-μm-radius metal wire was electrochemically etched to an ultrafine point with a nanometer-radius, the metal wire was electrochemically coated with a phenol–allyphenol copolymer film. The micrometer-sized electrode was fabricated by directly electrochemical coating the metal wire with an extremely thin phenol–allyphenol copolymer film. Then, the nanometer-radius electrode (the first electrode) and the 10-μm-radius electrode (the second electrode) were inserted into two sides of a thick-septum borosilicate theta (θ) tubing, respectively. The second electrode protruded from the top of the θ tubing. The top of the θ tubing was sealed with insulating ethyl α-cyanoacrylate. The top of the θ tubing with both electrodes was ground flat and polished successively with fine sandpaper and aluminum oxide powder until the tip of the first electrode was exposed. Since the second electrode protruded from the top of the θ tubing, its 10-μm-radius tip was naturally formed during polishing. The dual-disk electrodes were characterized by scanning electron microscopy and cyclic voltammetry. The success rate for fabrication of the dual-disk electrodes is ∼80% due to double insurance from two coating layers of different polymers.

We developed a simple fluorescence microscopy for acquisition of high-resolution images of single quantum dots (QDs) labeled to biomolecules on apical plasma membrane, in cell interior and on basal plasma membrane of living cells. The method was a combination of total internal reflection fluorescence microscopy (TIRFM) at apical cell surface and intracellular microscopy coupled with focusing objective. Insulin conjugated to single QD (insulin-QD) was chosen as the model system. In order to bind insulin-QDs to insulin receptors on the plasma membrane through the interaction between insulin and its receptor, as well as internalize them, the cells attached on a coverslip were incubated with biotinylated insulin and QD-streptavidin conjugate at 37 °C. Next, fluorescent molecules in the cells were photobleached by illuminating the cells using a 100-W mercury lamp with the wavelengths from 460 to 490 nm. Then, the incident angle of a laser beam was adjusted to produce total internal reflection at the apical surface of a single cell. In this case, the insulin-QDs in the whole cell were excited, and the fluorescent molecules outside the cell were not illuminated. Finally, the images of single insulin-QDs on the apical plasma membrane, in the cell interior and on the basal plasma membrane of the cell were taken by focusing the objective to different positions, respectively. The resolution and contrast of the fluorescent spots in the images were much higher than those obtained by using epi-fluorescence microscopy and comparable to those obtained by using the conventional TIRFM. The method improved the image acquisition speed for the images on the apical and basal plasma membrane using the conventional TIRFM, and could acquire the high-resolution images in the cell interior quickly.

A novel electrochemical method with a microfluidic device was developed for analysis of single cells. In this method, cell injection, loading and cell lysis, and electrokinetic transportation and detection of intracellular species were integrated in a microfluidic chip with a double-T injector coupled with an end-channel amperometric detector. A single cell was loaded at the double-T injector on the microfluidic chip by using electric field. Then, the docked cell was lysed by a direct current electric field strength of 220 V/cm. The analyte of interest inside the cell was electrokinetically transported to the detection end of separation channel and was electrochemically detected. External standardization was used to quantify the analyte of interest in individual cells. Ascorbic acid (AA) in single wheat callus cells was chosen as the model compound. AA could be directly detected at a carbon fiber disk bundle electrode. The selectivity of electrochemical detection made the electropherogram simple. The technique described here could, in principle, be applied to a variety of electroactive species within single cells.

Novel electrochemiluminescence resonance energy transfer (ECRET) between an emitter electrochemically generated by luminol as the donor and luminescent quantum dots as the acceptor is investigated. The ECRET technique can be used to study the interactions and conformational changes of proteins.