Secondary ion MS imaging of lipids in picoliter vials with a buckminsterfullerene ion source

Investigation of the spatial distribution of lipids in cell membranes can lead to an improved understanding of the role of lipids in biological function and disease. Time-of-flight secondary ion mass spectrometry is capable of molecule-specific imaging of biological molecules across single cells and has demonstrated potential for examining the functional segregation of lipids in cell membranes. In this paper, standard SIMS spectra are analyzed for phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, cholesterol, and sulfatide. Importantly, each of the lipids result in signature mass spectral peaks that allow them to be identified. These signature peaks are also useful for imaging experiments and are utilized here to simultaneously image lipids on a micrometer scale in picoliter vials. Because the low secondary ion signal achieved for lipids from an atomic primary ion source makes cell-imaging experiments challenging, improving signal with cluster primary ion sources is of interest. Here, we compare the secondary ion yield for seven lipids using atomic (Ga+ or In+) ion sources and a buckminsterfullerene (C60+) primary ion source. A 40-1000-fold improvement in signal is found with C60+ relative to the other two ion sources, indicating great promise for future cellular imaging applications using the C60+ probe.     

Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.     

Development of a microchamber array for picoliter PCR

A microchamber array for PCR was developed by semiconductor microfabrication technology. The microchambers were designed to be of picoliter quantity. To optimize fluid retention, the surface states of the substrate and the inner walls were examine for four different types of microchamber. The substrate was silicon, while silicon dioxide was selected for the inner walls. PCR was performed in the microchamber array, and the amplification of DNA was detected using a technique based on the energy transfer of fluorescent dyes. The lower volume limit for PCR was investigated using various sizes of microchambers. Microchambers with volume greater than 86 pL gave successful PCR. In addition, the system was improved in order to take up the PCR product. To prevent mixing of the samples, the samples were dried after PCR using a membrane that permeates only vapor.     

Possible binding sites for biotin stabilized water soluble Ag nanoparticles: an experimental and theoretical study

The evolution of Ag nanoparticles by photochemical reduction method and the effect of biotin on their UV-Visible absorption spectrum were studied. Surface modification studies were carried out on chemically reduced Ag nanoparticles. ATR-FTIR studies showed that the biotin molecules bind with the surface of Ag nanoparticles through the oxygen of the carboxylate group. Theoretical calculations were carried out on the structure of the biotin and the silver complex of biotin (biotin(-)-Ag+) by optimizing their structures using density functional calculations with the B3LYP method using the LANL2DZ basis set. Theoretical calculations and experimental evidence favors a preferential binding of biotin molecule to Ag nanoparticles through the carboxylate group.     

Spatially resolved detection of attomole quantities of organic molecules localized in picoliter vials using time-of-flight secondary ion mass spectrometry.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been utilized to detect femtomole and attomole quantities of organic species from within silicon nanovials. By using high-density arrays (10,000 nanovials/cm2) it is possible to chemically characterize diverse sample sets within a single chemical image. Molecular sensitivities, for the compounds investigated, very between 85 attomoles and 25 femtomoles, and typical acquisition times are approximately 100 ms per nanovial. These vials are fabricated using photolithography and KOH etching of Si[001] wafers to create wells, with a pyramidal cross section, ranging in size from 25 to 5625 micron 2. The volume ranges from 30 femtoliters to 100 picoliters, respectively. A drawn glass microinjector and solenoid-driven dispenser are utilized to array picoliter volumes of organic compounds into individual silicon nanovials. Solution concentrations typically range from 1 x 10(-2) to 1 x 10(-4) M allowing femtomole and even attomole quantities of material to be dispensed into each vial.     

Molecular imprinting of biotin derivatives and its application to competitive binding assay using nonisotopic labeled ligands

Synthetic biotin-binding polymers were prepared by molecular imprinting. Methacrylic acid (MAA) was copolymerized with ethylene glycol dimethacrylate in the presence of biotin methyl ester (B-Me) in chloroform. Hydrogen-bonding-based complexation of B-Me with MAA generates the binding sites complementary to B-Me after extracting B-Me from the resulting copolymers. Data from NMR titration suggest a one-to-one prepolymerization complex formation of B-Me with MAA in chloroform. A possible complex structure was estimated by docking of the most stable conformers by intermolecular Monte Carlo conformational search under the assumption of a one-to-one association. The selectivity of the imprinted polymers was investigated and an imprinted polymer-based competitive binding assay for B-Me was demonstrated using biotin p-nitrophenyl ester as a nonisotopic-labeled ligand.     

Magnetic levitation as a platform for competitive protein-ligand binding assays

This paper describes a method based on magnetic levitation (MagLev) that is capable of indirectly measuring the binding of unlabeled ligands to unlabeled protein. We demonstrate this method by measuring the affinity of unlabeled bovine carbonic anhydrase (BCA) for a variety of ligands (most of which are benzene sulfonamide derivatives). This method utilizes porous gel beads that are functionalized with a common aryl sulfonamide ligand. The beads are incubated with BCA and allowed to reach an equilibrium state in which the majority of the immobilized ligands are bound to BCA. Since the beads are less dense than the protein, protein binding to the bead increases the overall density of the bead. This change in density can be monitored using MagLev. Transferring the beads to a solution containing no protein creates a situation where net protein efflux from the bead is thermodynamically favorable. The rate at which protein leaves the bead for the solution can be calculated from the rate at which the levitation height of the bead changes. If another small molecule ligand of BCA is dissolved in the solution, the rate of protein efflux is accelerated significantly. This paper develops a reaction-diffusion (RD) model to explain both this observation, and the physical-organic chemistry that underlies it. Using this model, we calculate the dissociation constants of several unlabeled ligands from BCA, using plots of levitation height versus time. Notably, although this method requires no electricity, and only a single piece of inexpensive equipment, it can measure accurately the binding of unlabeled proteins to small molecules over a wide range of dissociation constants (K(d) values within the range from ~10 nM to 100 μM are measured easily). Assays performed using this method generally can be completed within a relatively short time period (20 min-2 h). A deficiency of this system is that it is not, in its present form, applicable to proteins with molecular weight greater than approximately 65 kDa.     

Silicon microfabricated column with microfabricated differential mobility spectrometer for GC analysis of volatile organic compounds

A 3.0-m-long, 150-microm-wide, 240-microm-deep channel etched in a 3.2-cm-square silicon chip, covered with a Pyrex wafer, and coated with a dimethyl polysiloxane stationary phase is used for the GC separation of volatile organic compounds. The column, which generates approximately 5500 theoretical plates, is temperature-programmed in a conventional convection oven. The column is connected through a heated transfer line to a microfabricated differential mobility spectrometer. The spectrometer incorporates a 63Ni source for atmospheric-pressure chemical ionization of the analytes. Nitrogen or air transport gas (flow 300 cm(3)/min) drives the analyte ions through the cell. The spectrometer operates with an asymmetric radio frequency (RF) electric field between a pair of electrodes in the detector cell. During each radio frequency cycle, the ion mobility alternates between a high-field and a low-field value (differential mobility). Ions oscillate between the electrodes, and only ions with an appropriate differential mobility reach a pair of biased collectors at the downstream end of the cell. A compensation voltage applied to one of the RF electrodes is scanned to allow ions with different differential mobilities to pass through the cell without being annihilated at the RF electrodes. A unique feature of the device is that both positive and negative ions are detected from a single experiment. The combined microfabricated column and detector is evaluated for the analysis of volatile organic compounds with a variety of functionalities.     

Fluorescence recovery assay for the detection of protein-DNA binding

We report a novel and straightforward fluorescence recovery assay which enables the detection of protein-DNA interactions and simultaneously determines relative binding affinities of sequence-specific DNA-binding proteins for a variety of DNA sequences in a multiplexed format. The detection of protein-DNA binding is accomplished by monitoring fluorescence recovery during exonuclease digestion of DNA sequences that are modified with fluorophore-quencher pairs. Retardation of fluorescence recovery occurs with binding of the protein to the putative DNA binding element, which arrests exonuclease digestion. The assay detects protein-DNA binding in a homogeneous solution simply, quickly, and reliably without using radioisotopes. Multiplexing is possible by labeling different DNA sequences with spectrally distinct dyes, allowing simultaneous analysis of experimental and control binding reactions in the same mixture.     

Competitive binding assay for glucose based on glycodendrimer-fluorophore conjugates

A new fluorescent glucose assay has been created using Alexa Fluor 647-labeled concanavalin A (Con A) and a fourth-generation PAMAM Alexa Fluor 594-labeled glycodendrimer. This assay has been shown to have a large response to glucose within the biological range and to be capable of functioning within a polymer hydrogel. In this paper, the glucose response is shown to be a single fluorophore-based quenching reaction. Data showing that the sensor is fully reversible and specific through competitive binding between the dendrimer and glucose with Con A are presented. Overall, the assay is shown to have potential over the traditional dextran-based assay because it has a larger dynamic response to physiological glucose concentrations, incorporates longer wavelength dyes that improve signal penetration through dermal tissue, and provides an internal reference in the form of a nonreactive fluorescent label.     

Characteristics of picoliter droplet dried residues as standards for direct analysis techniques

The characteristics of dried residues of picodroplets of single-, two-, and three-element aqueous solutions, which qualify these as reference materials in the direct analysis of single particles, single cells, and other microscopic objects using, e.g., laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOF-MS) and micro-X-ray fluorescence (MXRF), were evaluated. Different single-, two-, and three-element solutions (0.01-1 g/L) were prepared in picoliter volume (around 130 pL) with a thermal inkjet printing technique. An achievable dosing precision of 4-15% was calculated by total reflection X-ray fluorescence (TXRF) determination of the transferred elemental mass of an array of 100 droplets. The size of the dried residues was determined by optical microscopy to be 5-20 microm in diameter depending on the concentration and the surface material. The elemental distribution of the dried residues was determined with synchrotron micro-X-ray fluorescence (SR-MXRF) analyses. The MXRF results show high uniformity for element deposition of every single droplet with an RSTD of 4-6% depending on the concentration of spotted solution. The shape and height profile of dried residues from picoliter droplets were studied using atomic force microscopy (AFM). It was found that these dry to give symmetrical spherical segments with maximum heights of 1.7 microm. The potential of this technique for direct LA-ICP-TOF-MS analysis is shown.     

Development of quantitative cell-based enzyme assays in microdroplets

We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.     

Aptamer-based enantioselective competitive binding assay for the trace enantiomer detection

The development of highly enantioselective assays and sensors has received much attention for the determination of enantiomeric impurities at a low level. For chiral compounds, the efficient monitoring of the in selection procedure has allowed the isolation of nucleic acid aptamers which are able to strongly discriminate the target enantiomers. In this paper, we demonstrated for the first time that an aptamer can be successfully used to design a highly enantioselective tool for the trace enantiomer detection. The aptamer-based stereoselective assay was developed using an affinity capillary electrophoresis-based competitive, homogeneous format and an on-capillary mixing approach. Detection of as low as 0.01% of the minor enantiomer in a nonracemic mixture can be achieved, in a short analysis time (<5 min).     

Chemical transfection of cells in picoliter aqueous droplets in fluorocarbon oil

The manipulation of cells inside water-in-oil droplets is essential for high-throughput screening of cell-based assays using droplet microfluidics. Cell transfection inside droplets is a critical step involved in functional genomics studies that examine in situ functions of genes using the droplet platform. Conventional water-in-hydrocarbon oil droplets are not compatible with chemical transfection due to its damage to cell viability and extraction of organic transfection reagents from the aqueous phase. In this work, we studied chemical transfection of cells encapsulated in picoliter droplets in fluorocarbon oil. The use of fluorocarbon oil permitted high cell viability and little loss of the transfection reagent into the oil phase. We varied the incubation time inside droplets, the DNA concentration, and the droplet size. After optimization, we were able to achieve similar transfection efficiency in droplets to that in the bulk solution. Interestingly, the transfection efficiency increased with smaller droplets, suggesting effects from either the microscale confinement or the surface-to-volume ratio.     

Using liposomal fluorescent biolabels to develop an immunoaffinity chromatographic biosensing system for biotin

Although biotin is an important vitamin for cellular function and growth, there are no rapid, simple, and reliable analytical tools available for its quantitation in bodily fluids or foodstuffs. In this study, we developed an immunoaffinity chromatographic biosensing system for the direct determination of biotin. A stationary phase having affinity for biotin was synthesized by covalently bonding antibiotin monoclonal antibodies onto 90-microm, NHS-activated sepharose beads. The beads were then packed into 1.9-cm-diameter plastic tubes to form a column having a volume of 3.0 mL. The function of the proposed immunoaffinity chromatographic assay was based on competition between biotin and carboxyfluorescein (CF)-encapsulated, biotin-tagged liposomes (liposomal biolabels) for the limited number of antibiotin antibody binding sites. Buffers containing biotin standards at concentrations ranging from 10(-12) to 10(-3) M were passed through the column to trap and concentrate the biotin on the solid support. The unbound binding sites of the antibody were then occupied through subsequent addition of the liposomal biolabels. The addition of 35% methanol released the CF molecules from the lyzed bound liposomes; the fluorescence intensity of the released markers was then measured using a fluorometer. The calibration curve for biotin was linear over 8 orders of magnitude, from 10(-12) to 10(-4) M. The limit of detection of this immunoaffinity chromatographic biosensing system reached as low as 5.0 pg of biotin (equivalent to 500 microL of 4.10 x 10(-11) M biotin).     

Analysis in ultrasmall volumes: microdispensing of picoliter droplets and analysis without protection from evaporation

A new approach is reported for analysis of ultrasmall volumes. It takes advantage of the versatile positioning of a dispenser to shoot approximately 150-pL droplets of liquid onto a specific location of a substrate where analysis is performed rapidly, in a fraction of the time that it takes for the droplet to evaporate. In this report, the site where the liquid is dispensed carries out fast-scan cyclic voltammetry (FSCV), although the detection method does not need to be restricted to electrochemistry. The FSCV is performed at a microcavity having individually addressable gold electrodes, where one serves as working electrode and another as counter/pseudoreference electrode. Five or six droplets of 10 mM [Ru(NH(3))(6)]Cl(3) in 0.1 M KCl were dispensed and allowed to dry, followed by redissolution of the redox species and electrolyte with one or five droplets of water and immediate FSCV, demonstrating the ability to easily concentrate a sample and the reproducibility of redissolution, respectively. Because this approach does not integrate detection with microfluidics on the same chip, it simplifies fabrication of devices for analysis of ultrasmall volumes. It may be useful for single-step and multistep sample preparation, analyses, and bioassays in microarray formats if dispensing and changing of solutions are automated. However, care must be taken to avoid factors that affect the aim of the dispenser, such as drafts and clogging of the nozzle.     

Development of a new microtiter plate format for clinically relevant assays

A new format for the microtiter plate-based assays was proposed. The novelty involves the use of disk-shaped inserts for immobilization of biological and chemical reagents. The internal opening of the disks allows measurements of the reactions by standard microtiter plate readers without any additional steps involving liquid handling. Ideally the plate end-users just have to add the sample and take the measurement without any need of multiple reagent additions or transfer of the liquid to a different plate. The novel assay format also allows handling of reagents which are not soluble in an aqueous environment. As a proof of concept we describe here several model reactions which are compatible with microtiter plate format, such as monitoring enzymatic reactions catalyzed by glucose oxidase (GOx) and urease, measurements of proteins by BCA assay, analysis of pH, and concentration of antioxidants. The "mix and match" approach in the disk-shape format allows multiplexing and could be particularly useful for high throughput screening. One of the potential application areas for this novel assay format could be in a multianalyte system for measurement of clinically relevant analytes in primary care.     

Estrogen receptor binding assay method for endocrine disruptors using fluorescence polarization

A rapid, simple and nonhazardous assay method for endcrine disruptors was developed using an estrogen receptor (ER) and fluorescence polarization (FP). Among the fluorescent compounds, the 17alpha-fluorescein-labeled estradiol derivative was selected as the most suitable ligand for the ER binding assay, since it showed the highest affinity to ER. In the Scatchard plot analysis, its convex curve exhibited a positive cooperative binding, indicating the induction of a conformational change of the ER with the binding of the ligand to form a dimer and to increase the affinity for the additional ligand. On the basis of the Hill plot analysis, its dissociation constant and Hill coefficient were 10.4 nM and 1.63, respectively. A competitive binding assay with an unlabeled 17beta-estradiol (E2) yielded an IC50 value of 2.82 nM and a Hill coefficient of 1.67, thus providing a Ki value of 0.65 nM. In the same manner, the Hill coefficients for estrone, estriol, diethylstilbestrol, and tamoxifen were determined to be 0.99, 1.17, 1.59, and 2.44, respectively.