Single Particle Detection Research Articles

Detection of single magnetic bead for biological applications using an InAs quantum-well micro-Hall sensor

Room-temperature detection of a single commercial superparamagnetic bead (1.2μm in diameter) suitable for biological applications has been realized using an InAs quantum-well micro-Hall sensor. The detection was demonstrated using phase-sensitive detection on a single Hall cross as well as in a Hall gradiometry setup. The high signal to noise ratio, obtained in both configurations, promises detection of single nanometer-size particles by further miniaturization of the device to submicron dimensions.

Single particle detection and characterization of synuclein co-aggregation

Protein aggregation is the key event in a number of human diseases such as Alzheimer’s and Parkinson’s disease. We present a general method to quantify and characterize protein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT). In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes. As the ratio of two fluorescently labelled components can be analysed for each aggregate separately in a homogeneous assay, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. Using this method, we could show that wild-type α-synuclein forms co-aggregates with a mutant variant found in familial Parkinson’s disease. Moreover, we found a striking increase in aggregate formation at non-equimolar mixing ratios, which may have important therapeutic implications, as lowering the relative amount of aberrant protein may cause an increase of protein aggregation leading to adverse effects.

Ultra‐high sensitivity multi‐photon detection imaging in proteomics analyses

We report on the use of 125I and 131I labeling and of new, multicolor, multi-photon detection (MPD) methods to routinely and quantitatively detect protein spots on two-dimensional gel electrophoresis plates in the zeptomole to attomole range. We demonstrate that the MPD methodology can be used to detect radioactive labels on two-dimensional gels and has several characteristics that are advantageous for functional proteomics. First, by using single particle detectors, the sensitivity for detection of radiolabels can be improved dramatically. Second, because single particle detectors can differentiate the particle energies produced by different decay processes, it is possible to choose combinations of radioisotopes that can be detected and quantified individually on the same 2-D gel. Third, the MPD technology is essentially linear over six to seven orders of magnitude, i.e., it is possible to accurately quantify radiolabeled proteins over a range from at least 60 zeptomoles to 60 femtomoles. Finally for radionuclides that decay by electron capture, e.g., with emission of both beta and gamma rays, co-incident detection of two particles/photons can be used to detect such radionuclides well below background radiation levels. These methods are used to monitor acidic/phosphorylated proteins in as little as 60 ng of HeLa cells proteins.

Development of a secondary-electron ion-microscope for microbeam diagnostics

We describe a novel secondary-electron ion microscope (SEIM), designed for diagnostics of the upcoming sub-micron Columbia University charged-particle microbeam. This secondary-electron ion microscope allows much higher resolutions, at higher single particle detection efficiencies, than previously available, for rapid and accurate diagnostics of sub-micron charged-particle beams. Based on ion electron-emission microscopy (IEEM) and photo-electron microscopy (PEM), the SEIM involves conversion of the incident projectiles on a secondary-electron emitting film. The ejected electrons are focused using a unipolar electrostatic lens and conical electrostatic mirror to form a magnified image on a microchannel plate (MCP). The flight path of the electrons includes two 45° bends; this “folded” geometry results in lower aberrations than a “straight” design, and enables efficient beam imaging down to 100nm resolution with >50% single electron transfer efficiency.

Emission properties and applications of nanostructured luminescent oxide nanoparticles

Abstract Rare earth doped oxide materials are well known for their numerous applications in light emitting devices. An interesting issue is to study the emission properties of nanoparticles, with the aim to understand the influence of small size and surface effects on the emission processes. These particles could furthermore be used in new applications such as the elaboration of transparent emitting devices or new biological labels. The work presented here concerns highly luminescent rare earth doped yttrium vanadates (YVO 4 :Eu) and lanthanum phosphate LaPO 4 :Ce,Tb· x H 2 O nanoparticles. Simple aqueous colloidal syntheses are used for the elaboration of concentrated colloids based on the progressive decomposition of polymeric precursors at moderate temperature (60–90 °C). Both types of particles exhibit strong emission (quantum yields of 25% and 45% for vanadates and phosphates, respectively), but significantly lower than that for the equivalent bulk materials. The alteration of the emission processes is discussed in terms of surface quenching effects. Improvements are obtained through the elaboration of core/shell nanostructures. Surface derivatization has been achieved through the controlled growth of an organically modified silica shell using a functionalized silane precursor. Two examples are given concerning the applications of those particles. The first one is the elaboration of transparent and highly luminescent thin films, obtained by the dispersion of the functionalized particles in a sol–gel silica matrix. The other one is the use of guanidine functionalized particles as biological labels for the single particle detection of sodium channels in cardiac cells.

Feasibility of Detection and Identification of Individual Bioaerosols Using Laser-Induced Breakdown Spectroscopy

The detection and identification of individual bioaerosols using laser-induced breakdown spectroscopy (LIBS) is investigated using aerosolized Bacillus spores. Spores of Bacillus atrophaeous, Bacillus pumilus, and Bacillus stearothemophilus were introduced into an aerosol flow stream in a prescribed manner such that single-particle LIBS detection was realized. Bacillus spores were successfully detected based on the presence of the 393.4- and 396.9-nm calcium atomic emission lines. Statistical analyses based on the aerosol number density, the LIBS-based spore sampling frequency, and the distribution of the resulting calcium mass loadings support the conclusion of individual spore detection within single-shot laser-induced plasmas. The average mass loadings were in the range of 2-3 fg of calcium/Bacillus spore, which corresponds to a calcium mass percentage of approximately 0.5%. While individual spores were detected based on calcium emission, the resulting Bacillus spectra were free from CN emission bands, which has implications for the detection of elemental carbon, and LIBS-based detection of single spores based on the presence of magnesium or sodium atomic emission was unsuccessful. Based on the current instrumental setup and analyses, real-time LIBS-based detection and identification of single Bacillus spores in ambient (i.e., real life) conditions appears unfeasible.

A fast low-noise charged-particle CVD diamond detector

This work presents the design and test results of a charged-particle solid-state detector with ultrafast signal response based on polycrystalline chemical-vapor-deposition (pCVD) diamond as active detector material and a high-bandwidth RF amplifier. We tested the detector at the Indiana University Cyclotron Facility Bloomington, IN, in a proton beam with a kinetic energy ranging from 55 to 200 MeV. The detector signals showed an average pulsewidth of 1.38 ns, which enables single-particle counting at instantaneous rates approaching the gigahertz range. The detector operated with a signal-to-noise ratio of 7 : 1 for 200-MeV protons and a single-particle detection efficiency up to 99%.

Electrical detection of single viruses.

We report direct, real-time electrical detection of single virus particles with high selectivity by using nanowire field effect transistors. Measurements made with nanowire arrays modified with antibodies for influenza A showed discrete conductance changes characteristic of binding and unbinding in the presence of influenza A but not paramyxovirus or adenovirus. Simultaneous electrical and optical measurements using fluorescently labeled influenza A were used to demonstrate conclusively that the conductance changes correspond to binding/unbinding of single viruses at the surface of nanowire devices. pH-dependent studies further show that the detection mechanism is caused by a field effect, and that the nanowire devices can be used to determine rapidly isoelectric points and variations in receptor-virus binding kinetics for different conditions. Lastly, studies of nanowire devices modified with antibodies specific for either influenza or adenovirus show that multiple viruses can be selectively detected in parallel. The possibility of large-scale integration of these nanowire devices suggests potential for simultaneous detection of a large number of distinct viral threats at the single virus level.

MRI detection of single particles for cellular imaging.

There is rapid growth in the use of MRI for molecular and cellular imaging. Much of this work relies on the high relaxivity of nanometer-sized, ultrasmall dextran-coated iron oxide particles. Typically, millions of dextran-coated ultrasmall iron oxide particles must be loaded into cells for efficient detection. Here we show that single, micrometer-sized iron oxide particles (MPIOs) can be detected by MRI in vitro in agarose samples, in cultured cells, and in mouse embryos. Experiments studying effects of MRI resolution and particle size from 0.76 to 1.63 microm indicated that T(2)* effects can be readily detected from single MPIOs at 50-microm resolution and significant signal effects could be detected at resolutions as low as 200 microm. Cultured cells were labeled with fluorescent MPIOs such that single particles were present in individual cells. These single particles in single cells could be detected both by MRI and fluorescence microscopy. Finally, single particles injected into single-cell-stage mouse embryos could be detected at embryonic day 11.5, demonstrating that even after many cell divisions, daughter cells still carry individual particles. These results demonstrate that MRI can detect single particles and indicate that single-particle detection will be useful for cellular imaging.

Multiplexed Electrical Detection of Single Viruses

ABSTRACTWe report direct, real-time electrical detection of single virus particles with high selectivity using nanowire field effect transistors. Measurements made with nanowire arrays modified with antibodies for influenza A showed discrete conductance changes characteristic of binding and unbinding in the presence of influenza A but not paramyxovirus or adenovirus. Moreover, simultaneous electrical and optical measurements using fluorescently-labelled influenza A demonstrate conclusively that the conductance changes correspond to binding/unbinding of single viruses at the surface of nanowire devices. In addition, studies of nanowire devices modified with antibodies specific for either influenza or adenovirus show that multiple viruses can be selectively detected in parallel. The possibility of large scale integration of these nanowire devices suggests potential for simultaneous detection of a large number of distinct viral threats at the single virus level.

Low-temperature detectors

Low-temperature detectors offer single particle detection with high-energy resolution and low-energy threshold, what is of interest for applications both in fundamental physics and industry. Different detector concepts as well as existing/planned applications and refrigeration techniques are presented.

Shift of whispering-gallery modes in microspheres by protein adsorption.

Biosensors based on the shift of whispering-gallery modes in microspheres accompanying protein adsorption are described by use of a perturbation theory. For random spatial adsorption, theory predicts that the shift should be inversely proportional to microsphere radius R and proportional to protein surface density and excess polarizability. Measurements are found to be consistent with the theory, and the correspondence enables the average surface area occupied by a single protein to be estimated. These results are consistent with crystallographic data for bovine serum albumin. The theoretical shift for adsorption of a single protein is found to be extremely sensitive to the target region, with adsorption in the most sensitive region varying as 1/R(5/2). Specific parameters for single protein or virus particle detection are predicted.

Ion-induced emission microscopies

Abstract New emission-based MeV nuclear microscopies have been in the process of development for the past four years. These techniques all fall under the heading of ion-induced emission microscopy (I-IEM), and the first to be developed was ion-electron emission microscopy (IEEM). With I-IEM the ion beam is not focused, but instead, secondary particles emitted when a single-ion strikes the sample are projected at great magnification onto a high efficiency single particle detector generating position signals. These X and Y signals are then put into coincidence with other signals made by this same ion in a fashion completely analogous to traditional nuclear microprobe analysis. In this paper, we update the current state of I-IEMs, which currently includes IEEM and highly charged ion-secondary ion mass spectroscopy (HCI-SIMS or IIEM) and ion-photon emission microscopy. At the present time none of these microscopies have atomic resolution, but the potential exists for resolution adequate for many scientific and nanotechnology applications.

Gas phase electrochemical detection of single latex particles.

In this study we describe zero current potentiometric measurements in a gaseous flame electrolyte, for the detection of single latex particles. Combustion of polystyrene latex particles when added to a premixed hydrogen/oxygen/nitrogen flame, results in an increase in charged species relative to the surrounding hydrogen flame. As a consequence of this increase in ionic concentration over background, short-lived potential difference transients were measured between two platinum indicator electrodes placed in a two-compartment flame electrochemical cell (described in Electrochem. Commun., 2001, 3, 675-681). The frequency of the transient events was dependent on the number density of latex particles in solution. It is proposed that each short-lived transient event corresponds to the combustion of single latex particles in a flame. A potential difference maximum of 0.56 V when 3.0 microm diameter particles were added to the flame was measured. Also it was shown that it is possible to detect 0.3 microm diameter latex particles using the same technique. It is postulated that the physical basis of the potential difference is due to the establishment of diffusion/junction potential due to the increase in ionisation from polystyrene combustion at the surface of one indicator electrode. This methodology may be applied to the detection of particulates composed of ionisable species (organic or inorganic) in gaseous environment such as bacteria, viruses, pollen grains and dust.

Development of Vertically Integrated Imaging and Particle Sensors

AbstractIntegrated imaging and particle sensors have been developed using thin-film on ASIC technology. For this purpose, hydrogenated amorphous silicon diodes, in various configurations, have been optimized for imaging and direct particle detection. These devices were first deposited on glass substrates and later on CMOS readout chips. With an optimization of the material properties and of the diode, a dark current of 1 pA/cm2 could be achieved on p-i-n structures at reverse bias voltage of 1 V. CMOS imagers, incorporating these optimized diodes were then fabricated and characterized. Very thick diodes (with thicknesses up to 50 μm) were also optimized and deposited on glass and on CMOS readout chips. Particle detectors in TFA technology with 12 and 30 μm a-Si:H n-i-p diodes have been fabricated and characterized using light pulse illumination. Direct detection of single low-energy beta particles has been demonstrated.

Silicon Micro-Photonic Structure for Ultra-Sensitive Biosensing

ABSTRACTWe demonstrate a micron-size planar silicon photonic device that is able to detect low concentrations of metal nano-particles approaching single particle detection. This sensitivity is achieved by using strong light confining structures that enhance the extinction cross-section of metal nano-particles by orders of magnitude. Structures were fabricated and measurements of the transmission spectra of the devices demonstrate the detection of 10 nm diameter gold particles resting on the device with a density of fewer than 2 particles per 104 nm2 (the area of the sensing region surface). Using such a device, in a fluidic platform, one could detect the presence of a single metal nano-particle specifically bound to various analytes, enabling ultrasensitive detection of analytes including DNA, RNA, proteins, and antigens.

Detection and enumeration of single nanometric particles: A confocal optical design for fluorescence flow cytometry

Nanometer-sized particles are increasingly important in fields ranging from technology development to environmental analysis. Traditionally, quantification of biological nanometric particles, such as viruses, has been challenging. Recent advances in optics and optical design have made single molecule detection possible in flowing systems. However, many commercially available flow cytometers are not optimized for routine analysis of ultrasmall biological particles. In this work, a confocal optical arrangement was incorporated into the design of a relatively simple flow cytometer optimized for rapid enumeration of fluorescent nanometric particles. The instrument was designated the “single nanometric particle enumerator” (SNaPE). Instrument parameters, such as data acquisition rates, flow rates, and minimum sampling time were examined and optimized for the SNaPE. The measured detection efficiency was ∼8%, which is consistent with the confocal geometry and equivalent to the detection efficiency reported in many single molecule studies. Signal calibration was achieved using fluorescent polystyrene spheres ranging from 26 nm to 10 μm in diameter. The SNaPE exhibited a limit of detection of ∼180 fluorescein isothiocyanate equivalents and was capable of particle enumeration over the tested concentration range of 106–109 particles/ml.

A broad-application microchannel-plate detector system for advanced particle or photon detection tasks: large area imaging, precise multi-hit timing information and high detection rate

New applications for single particle and photon detection in many fields require both large area imaging performance and precise time information on each detected particle. Moreover, a very high data acquisition rate is desirable for most applications and eventually the detection and imaging of more than one particle arriving within a microsecond is required. Commercial CCD systems lack the timing information whereas other electronic microchannel plate (MCP) read-out schemes usually suffer from a low acquisition rate and complicated and sometimes costly read-out electronics. We have designed and tested a complete imaging system consisting of an MCP position readout with helical wire delay-lines, single-unit amplifier box and PC-controlled time-to-digital converter (TDC) readout. The system is very flexible and can detect and analyse position and timing information at single particle rates beyond 1 MHz. Alternatively, multi-hit events can be collected and analysed at about 20 kHz rate. We discuss the advantages and applications of this technique and then focus on the detector's ability to detect and analyse multiple hits.

The radiation environment in and near high gradient rf cavities

Measurements of the X-ray flux, electron flux and spectrum are presented with the aim of evaluating the usefulness of ionization sensitive single particle detectors in the immediate environment of an rf cavity. The measured fluxes are found to be roughly consistent with field emission of electrons followed by one generation of bremsstrahlung photon production, with absorption and scattering of the final photons and electrons in the cavity and vacuum chamber structure.

Real-time measurements of the chemical composition of size-resolved particles during a Santa Ana wind episode, California USA

Size-resolved particle composition, mass and number concentrations, aerosol scattering coefficients, and prevailing meteorological conditions were measured at the Ellen Browning Scripps Memorial Pier located in La Jolla, California on 15 December 1998. Aerosol particles were sampled using a field transportable aerosol time-of-flight mass spectrometer, allowing for the continuous detection and characterization of single particles from a polydisperse sample. An extensive and rapid change in the chemical composition of aerosol particles with aerodynamic diameters between 1.0 and 2.5 μm has been observed during the onset of a Santa Ana Winds condition. Coincident with the observed change in meteorological conditions, a substantial decrease in sea salt particles corresponds to an increase in dust and carbon-containing particles. This paper examines observations of the rapid changes occurring in the chemical composition of single aerosol particles and demonstrates the new types of information that can be obtained by measuring single particle size and composition with high temporal resolution.