Shaobin Zhu

Light-Scattering Detection below the Level of Single Fluorescent Molecules for High-Resolution Characterization of Functional Nanoparticles

Ultrasensitive detection and characterization of single nanoparticles (<100 nm) is important in nanotechnology and life sciences. Direct measurement of the elastically scattered light from individual nanoparticles represents the simplest and the most direct method for particle detection. However, the sixth-power dependence of scattering intensity on particle size renders very small particles indistinguishable from the background. Adopting strategies for single-molecule fluorescence detection in a sheathed flow, here we report the development of high sensitivity flow cytometry (HSFCM) that achieves real-time light-scattering detection of single silica and gold nanoparticles as small as 24 and 7 nm in diameter, respectively. This unprecedented sensitivity enables high-resolution sizing of single nanoparticles directly based on their scattered intensity. With a resolution comparable to that of TEM and the ease and speed of flow cytometric analysis, HSFCM is particularly suitable for nanoparticle size distribution analysis of polydisperse/heterogeneous/mixed samples. Through concurrent fluorescence detection, simultaneous insights into the size and payload variations of engineered nanoparticles are demonstrated with two forms of clinical nanomedicine. By offering quantitative multiparameter analysis of single nanoparticles in liquid suspensions at a throughput of up to 10 000 particles per minute, HSFCM represents a major advance both in light-scattering detection technology and in nanoparticle characterization.

Size Differentiation and Absolute Quantification of Gold Nanoparticles via Single Particle Detection with a Laboratory-Built High-Sensitivity Flow Cytometer

Employing single nanoparticle detection with a laboratory-built high-sensitivity flow cytometer, we developed a simple and versatile platform that is capable of detecting the surface plasmon resonance scattering of gold nanoparticles (GNPs) as small as 24 nm, differentiating GNPs of different sizes, and providing accurate quantification of GNPs. Low-concentration samples (fM to pM) in small volumes (microL) can be measured in minutes with an analysis rate of up to 100-200 GNPs per second. Among these features, absolute quantification provides a distinct advantage because it does not require standard samples.

Rapid, absolute, and simultaneous quantification of specific pathogenic strain and total bacterial cells using an ultrasensitive dual-color flow cytometer.

This paper describes a rapid and sensitive strategy for the absolute and simultaneous quantification of specific pathogenic strain and total bacterial cells in a mixture. A laboratory-built compact, high-sensitivity, dual channel flow cytometer (HSDCFCM) was modified to enable dual fluorescence detection. A bacterial cell mixture comprising heat-killed pathogenic Escherichia coli E. coli O157:H7 and harmless E. coli DH5alpha was used as a model system. Pathogenic E. coli O157:H7 cells were selectively labeled by red fluorescent probe via antibody-antigen interaction, and all bacterial cells were stained with membrane-permeable nucleic acid dye that fluoresces green. When each individual bacterium passes through the interrogating laser beam, E. coli O157:H7 emits both red and green fluorescence, while E. coli DH5alpha exhibits only green fluorescence. Because the fluorescence burst generated from each individual bacterial cell was easily distinguished from the background, accurate enumeration and consequently absolute quantification were achieved for both pathogenic and total bacterial cells. By using this strategy, accurate counting of bacteria at a density above 1.0 x 10(5) cells/mL can be accomplished with 1 min of data acquisition time after fluorescent staining. Excellent correlation between the concentrations measured by the HSDCFCM and the conventional plate-counting method were obtained for pure-cultured E. coli O157:H7 (R(2) = 0.9993) and E. coli DH5alpha (R(2) = 0.9998). Bacterial cell mixtures with varying proportions of E. coli O157:H7 and E. coli DH5alpha were measured with good ratio correspondence. We applied the established approach to detecting artificially contaminated drinking water samples; E. coli O157:H7 of 1.0 x 10(2) cells/mL were accurately quantified upon sample enrichment. It is believed that the proposed method will find wide applications in many fields demanding bacterial identification and quantification.

Electrochemical intramolecular aminooxygenation of unactivated alkenes.

An electrochemical approach to the intramolecular aminooxygenation of unactivated alkenes has been developed. This process is based on the addition of nitrogen-centered radicals, generated through electrochemical oxidation, to alkenes followed by trapping of the cyclized radical intermediate with 2,2,6,6-tetramethylpiperidine-N-oxyl radical (TEMPO). Difunctionalization of a variety of alkenes with easily available carbamates/amides and TEMPO affords aminooxygenation products in high yields and with excellent trans selectivity for cyclic systems (d.r. up to>20:1). The approach provides a much-needed complementary route to existing cis-selective methods.

Development of an Ultrasensitive Dual-Channel Flow Cytometer for the Individual Analysis of Nanosized Particles and Biomolecules

A compact, high-sensitivity, dual-channel flow cytometer (HSDCFCM) was developed for the individual analysis of nanosized particles and biomolecules. A hydrodynamic focusing technique was applied to confine the sample stream and enable small probe volume. Fluorescence bursts from single R-phycoerythrin (R-PE) molecules passing through the laser beam were well resolved from the background with signal-to-noise ratio of 17. Excellent size discrimination was demonstrated with a mixture of three sizes of polystyrene nanoparticles. Simultaneous measurement of fluorescence and light scattering signals from individual nanoparticles was demonstrated with the 100 nm fluorescent latex beads. Doxorubicin-loaded ZrO(2) nanoparticles and fluorescently stained Escherichia coli ER2738 cells were analyzed successfully with dual-channel detection. Particle counting is demonstrated with the 210 nm fluorescent latex beads, and excellent correlation (R(2) > 0.998) between the manufacturer-reported concentrations and those measured by HSDCFCM enumeration was obtained. The measured sample detection efficiency was approximately 90% on average for particle concentrations ranging from 1.62 x 10(5) to 3.93 x 10(7) particles/mL. Sample mixtures with varying proportions of fluorescently labeled and unlabeled nanoparticles were also analyzed with good ratio correspondence. By providing rapid, quantitative, and multiparameter characterization of nanoparticles, it is believed that the HSDCFCM will find many applications in the fields of bionanotechnology, bioanalytical chemistry, and biomedicine.

Detection and quantification of bacterial autofluorescence at the single-cell level by a laboratory-built high-sensitivity flow cytometer

Cellular autofluorescence can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with or even precluding the detection of low-level specific fluorescence. Here we developed a method to detect and quantify bacterial autofluorescence in the green region of the spectrum at the single-cell level using a laboratory-built high-sensitivity flow cytometer (HSFCM). The detection of the very weak bacterial autofluorescence was confirmed by analyzing polystyrene beads of comparable and larger size than bacteria in parallel. Dithionite reduction and air re-exposure experiments verified that the green autofluorescence mainly originates from endogenous flavins. Bacterial autofluorescence was quantified by calibrating the fluorescence intensity of nanospheres with known FITC equivalents, and autofluorescence distribution was generated by analyzing thousands of bacterial cells in 1 min. Among the eight bacterial strains tested, it was found that bacterial autofluorescence can vary from 80 to 1400 FITC equivalents per cell, depending on the bacterial species, and a relatively large cell-to-cell variation in autofluorescence intensity was observed. Quantitative measurements of bacterial autofluorescence provide a reference for the background signals that can be expected with bacteria, which is important in guiding studies of low-level gene expression and for the detection of low-abundance biological molecules in individual bacterial cells. This paper presents the first quantification of bacterial autofluorescence in FITC equivalents.

Sensitive and Selective Bacterial Detection Using Tetracysteine-Tagged Phages in Conjunction with Biarsenical Dye†

National Natural Science Foundation of China[20675070, 20975087, 90913015, 21027010]; Program for New Century Excellent Talents in University[NCET-07-0729]; Research Funds for the Doctoral Program of Higher Education of China[20090121120008, 20090121110009]; NFFTBS[J1030415]

High-Throughput Multiparameter Analysis of Individual Mitochondria

Mitochondria are one of the most important organelles responsible for cellular energy metabolism and apoptosis regulation. However, single-mitochondrion analysis is challenging, because of their small sizes and the low content of organelle constituents. Here, we report the development of a sensitive and versatile platform for high-throughput multiparameter analysis of individual mitochondria. Employing specific fluorescent staining with a laboratory-built high-sensitivity flow cytometer (HSFCM), we demonstrate the simultaneous detection of side scatter, cardiolipin, and mitochondria DNA (mtDNA) of a single mitochondrion. Simultaneous measurements of side scatter, porin, and cytochrome c of individual mitochondria are reported for the first time. Correlation analysis among multiple attributes on an organelle-by-organelle basis could provide a more definitive assessment of the purity, structure integrity, and apoptosis-related proteins of isolated mitochondria than bulk measurement. This work represents a significant advancement in single-mitochondrion analysis. We believe that the HSFCM holds great potential for studying apoptotic signal transduction pathways at the single-mitochondrion level.

Identification of Mitochondria-Targeting Anticancer Compounds by an in Vitro Strategy

Mitochondria play a pivotal role in determining the point-of-no-return of the apoptotic process. Therefore, anticancer drugs that directly target mitochondria hold great potential to evade resistance mechanisms that have developed toward conventional chemotherapeutics. In this study, we report the development of an in vitro strategy to quickly identify the therapeutic agents that induce apoptosis via directly affecting mitochondria. This result is achieved by treating isolated mitochondria with potential anticancer compounds, followed by simultaneously measuring the side scatter and mitochondrial membrane potential (Δψ(m)) fluorescence of individual mitochondria using a laboratory-built high-sensitivity flow cytometer. The feasibility of this method was tested with eight widely used anticarcinogens. Dose-dependent Δψ(m) losses were observed for paclitaxel, antimycin A, betulinic acid, curcumin, ABT-737, and triptolide, but not for cisplatin or actinomycin D, which agrees well with their mechanisms of apoptosis induction reported in the literature. The as-developed method offers an effective approach to identify mitochondria-targeting anticancer compounds.

Analytical techniques for single-liposome characterization

Liposomes or phospholipid vesicles are one of the most versatile nanoparticles used to convey drugs, vaccines, genes, enzymes, or other substances to target cells and as a model to mimic biological membranes. To fulfil their roles in drug delivery and biotechnology, the physical and chemical properties of liposomes, such as size, shape, chemical composition, lamellarity, encapsulation efficiency of cargo molecules, and the density of proteins reconstituted in the membrane, need to be characterized to ensure reproducible preparation of the vesicles. Compared to bulk analysis, techniques focusing on the individual analysis of liposomes can reveal heterogeneity that is otherwise masked by ensemble averaging. Herein, we review the recent advances in techniques for single-liposome characterization.