Toward label-free optical fractionation of blood--optical force measurements of blood cells

There is a compelling need to develop systems capable of processing blood and other particle streams for detection of pathogens that are sensitive, selective, automated, and cost/size effective. Our research seeks to develop laser-based separations that do not rely on prior knowledge, antibodies, or fluorescent molecules for pathogen detection. Rather, we aim to harness inherent differences in optical pressure, which arise from variations in particle size, shape, refractive index, or morphology, as a means of separating and characterizing particles. Our method for measuring optical pressure involves focusing a laser into a fluid flowing opposite to the direction of laser propagation. As microscopic particles in the flow path encounter the beam, they are trapped axially along the beam and are pushed upstream from the laser focal point to rest at a point where the optical and fluid forces on the particle balance. On the basis of the flow rate at which this balance occurs, the optical pressure felt by the particle can be calculated. As a first step in the development of a label-free device for processing blood, a system has been developed to measure optical pressure differences between the components of human blood, including erythrocytes, monocytes, granulocytes, and lymphocytes. Force differentials have been measured between various components, indicating the potential for laser-based separation of blood components based upon differences in optical pressure. Potential future applications include the early detection of blood-borne pathogens for the prevention of sepsis and other diseases as well as the detection of biological threat agents.     

Photophoretic velocimetry for the characterization of aerosols

Aerosols are particles in a size range from some nanometers to some micrometers suspended in air or other gases. Their relevance varies as wide as their origin and composition. In the earth's atmosphere they influence the global radiation balance and human health. Artificially produced aerosols are applied, e.g., for drug administration, as paint and print pigments, or in rubber tire production. In all these fields, an exact characterization of single particles as well as of the particle ensemble is essential. Beyond characterization, continuous separation is often required. State-of-the-art separation techniques are based on electrical, thermal, or flow fields. In this work we present an approach to apply light in the form of photophoretic (PP) forces for characterization and separation of aerosol particles according to their optical properties. Such separation technique would allow, e.g., the separation of organic from inorganic particles of the same aerodynamic size. We present a system which automatically records velocities induced by PP forces and does a statistical evaluation in order to characterize the particle ensemble properties. The experimental system essentially consists of a flow cell with rectangular cross section (1 cm(2), length 7 cm), where the aerosol stream is pumped through in the vertical direction at ambient pressure. In the cell, a laser beam is directed orthogonally to the particle flow direction, which results in a lateral displacement of the particles. In an alternative configuration, the beam is directed in the opposite direction to the aerosol flow; hence, the particles are slowed down by the PP force. In any case, the photophoretically induced variations of speed and position are visualized by a second laser illumination and a camera system, feeding a mathematical particle tracking algorithm. The light source inducing the PP force is a diode laser (lambda = 806 nm, P = 0.5 W).     

Phage-Based Magnetoelastic Wireless Biosensors for Detecting Bacillus Anthracis Spores

A biosensor for the detection of biological warfare agents (Bacillus anthracis spores) was developed that combines the phage display technique with a magnetoelastic wireless detection platform. The affinity-based biosensor utilizes a phage-derived diagnostic probe as the biomolecular recognition element to capture target agents multivalently. Upon binding of the target agent to the sensor surface, the resonance frequency of the magnetoelastic biosensors decreases due to the additional mass of the target agent. Scanning electron microscopy was used to confirm binding of spores to the sensor surface. The sensitivity of the magnetoelastic acoustic sensor was tested to be 130 Hz per order of magnitude of spore concentration with a detection limit of 10³ spores/ml. The specificity of the sensors was tested against spores of other closely related Bacillus species and a large preferential binding to Bacillus anthracis spores was observed. The longevity of the phage based biosensor was compared to traditional antibody based biosensors and found to exhibit a much longer life     

Photocatalytic inactivation of spores of Bacillus anthracis using titania nanomaterials

Studies on photocatalytic inactivation of spores of Bacillus anthracis have been carried out using nanosized titania materials and UVA light or sun light. Results demonstrated pseudo first order behaviour of spore inactivation kinetics. The value of kinetic rate constant increased from 0.4h(-1) to 1.4h(-1) indicating photocatalysis facilitated by addition of nanosized titania. Nanosized titania exhibited superior inactivation kinetics on par with large sized titania. The value of kinetic rate constant increased from 0.02 h(-1) to 0.26 h(-1) on reduction of size from 1000 nm to 16 nm depicting the enhanced rate of inactivation of Bacillus anthracis Sterne spores on the decrease of particle size.     

Method of measuring Bacillus anthracis spores in the presence of copious amounts of Bacillus thuringiensis and Bacillus cereus

A sensitive and reliable method for the detection of Bacillus anthracis (BA; Sterne strain 7702) spores in presence of large amounts of Bacillus thuringiensis (BT) and Bacillus cereus (BC) is presented based on a novel PZT-anchored piezoelectric excited millimeter-sized cantilever (PAPEMC) sensor with a sensing area of 1.5 mm2. Antibody (anti-BA) specific to BA spores was immobilized on the sensing area and exposed to various samples of BA, BT, and BC containing the same concentration of BA at 333 spores/mL, and the concentration of BT + BC was varied in concentration ratios of (BA:BT + BC) 0:1, 1:0, 1:1, 1:10, 1:100, and 1:1000. In each case, the sensor responded with an exponential decrease in resonant frequency and the steady-state frequency changes reached were 14 +/- 31 (n = 11), 2742 +/- 38 (n = 3), 3053 +/- 19 (n = 2), 2777 +/- 26 (n = 2), 2953 +/- 24 (n = 2), and 3105 +/- 27 (n = 2) Hz, respectively, in 0, 27, 45, 63, 154, and 219 min. The bound BA spores were released in each experiment, and the sensor response was nearly identical to the frequency change during attachment. These results suggest that the transport of BA spores to the antibody immobilized surface was hindered by the presence of other Bacillus species. The observed binding rate constant, based on the Langmuir kinetic model, was determined to be 0.15 min-1. A hindrance factor (alpha) is defined to describe the reduced attachment rate in the presence of BT + BC and found to increase exponentially with BT and BC concentration. The hindrance factor increased from 3.52 at 333 BT + BC spores/mL to 11.04 at 3.33 x 105 BT + BC spores/mL, suggesting that alpha is a strong function of BT and BC concentration. The significance of these results is that anti-BA functionalized PEMC sensors are highly selective to Bacillus anthracis spores and the presence of other Bacillus species, in large amounts, does not prevent binding but impedes BA transport to the sensor.     

Concentration of Bacillus spores by using silica magnetic particles

Silica particles are mainly used for the concentration of nucleic acid for diagnostic purposes. This is usually done under acidic or chaotropic conditions that will demolish most of the living organisms and prevent the application of other diagnostic tests. Here we describe the development of a method for the capturing and concentration of Bacillus spores using silica magnetic particles to enable fast and sensitive detection. We have shown that capturing various Bacilli spores via silica magnetic particles is limited, with large differences between spore batches (42 +/- 25%). The hydrophobic exosporium layer of spore limits the capture by the hydrophilic silica beads. Partial removal of Bacillus exosporium increases capture efficiency. To increase capturing efficiency without harming the spores' viability, a cationic lipid, didecyldimethylammonium bromide (DDAB), was used as a coat for the negatively charged silica particles. DDAB treatment increased capture efficiency from 42% to more than 90%. Using this method, we were able to capture as few as 100 Bacillus anthracis spores/mL with 90% efficacy. Release of captured spores was achieved by the addition of albumin. The capture and release processes were verified by plating and by flow cytometry using light scatter analysis. The method is simple, efficient, easy to operate, and fast.     

A miniature biochip system for detection of aerosolized Bacillus globigii spores

The feasibility of using a novel detection scheme for the analysis of biological warfare agents is demonstrated using Bacillus globigii spores, a surrogate species for Bacillus anthracis. In this paper, a sensitive and selective enzyme-linked immunosorbent assay using a novel fluorogenic alkaline phosphatase substrate (dimethylacridinone phosphate) is combined with a compact biochip detection system, which includes a miniature diode laser for excitation. Detection of aerosolized spores was achieved by coupling the miniature system to a portable bioaerosol sampler, and the performance of the antibody-based recognition and enzyme amplification method was evaluated. The bioassay performance was found to be compatible with the air sampling device, and the enzymatic amplification was found to be an attractive amplification method for detection of low spore concentrations. The combined portable bioaerosol sampler and miniature biochip system detected 100 B. globigii spores, corresponding to 17 aerosolized spores/L of air. Moreover, the incorporation of the miniature diode laser with the self-contained biochip design allows for a compact system that is readily adaptable to field use. In addition, these studies have included investigations into the tradeoff between assay time and sensitivity.     

Cross-type optical particle separation in a microchannel

A continuous, real-time optical particle separation, which was previously delineated theoretically, is successfully implemented experimentally for the first time. In this method, particles suspended in a flowing fluid are irradiated with a laser beam propagating in a direction perpendicular to direction of fluid flow. Upstream of the laser beam, the particles move parallel to the direction of fluid flow. When the particles pass through the laser beam, the scattering force pushes them in the direction of laser beam propagation, causing the particles to be displaced perpendicular to the fluid flow direction. This displacement, known as the retention distance, depends on the particle size and the laser beam parameters. Finally, the particles escape from the laser beam and maintain their retention distances as they move downstream. In the present work, the trajectories and retention distances of polystyrene latex microspheres with three distinct diameters were monitored and measured using cross-type optical particle separation. The measured retention distances for different-sized particles were in good agreement with theoretical predictions.     

Characterization of Bacillus spore species and their mixtures using postsource decay with a curved-field reflectron

A strategy is proposed for the rapid identification of Bacillus spores, which relies on the selective release of a family of proteins, referred to as small, acid-soluble spore proteins (SASPs). In this work, SASPs were selectively solubilized from Bacillus spores on the MALDI sample plate by using 10% TFA. Proteolytic digests of SASPs generated in situ from spores of B. subtilis 168, B. globigii, B. thuringiensis subs. Kurstaki HD-1, B. cereus T, and the nonpathogenic strain B. anthracis Sterne were prepared in 5-25 min by using trypsin immobilized on Agarose beads and subsequently analyzed by MALDI-TOFMS using a curved-field reflectron. Protein identification was obtained by partial sequencing of distinctive tryptic peptides from Bacillus spores via post-source decay analysis combined with genome-based database searches by Mascot Sequence Query. Various unique SASPs were identified, allowing the characterization of Bacillus species by obtaining sequence-specific information on single peptides. The applicability of this approach for the rapid identification of Bacillus species was further established by analyzing spore mixtures.     

Interplay between Short‐ and Long‐Ranged Forces Leading to the Formation of Ag Nanoparticle Superlattice

Nanoparticle (NP) superlattices have attracted increasing attention due to their unique physicochemical properties. However, key questions persist regarding the correlation between short‐ and long‐range driving forces for nanoparticle assembly and resultant capability to predict the transient and final superlattice structure. Here the self‐assembly of Ag NPs in aqueous solutions is investigated by employing in situ liquid cell transmission electron microscopy, combined with atomic force microscopy‐based force measurements, and theoretical calculations. Despite the NPs exhibiting instantaneous Brownian motion, it is found that the dynamic behavior of NPs is correlated with the van der Waals force, sometimes unexpectedly over relatively large particle separations. After the NPs assemble into clusters, a delicate balance between the hydration and van der Waals forces results in a distinct distribution of particle separation, which is ascribed to layers of hydrated ions adsorbed on the NP surface. The study demonstrates pivotal roles of the complicated correlation between interparticle forces; potentially enabling the control of particle separation, which is critical for tailoring the properties of NP superlattices.     

Meniscus and viscous forces during normal separation of liquid-mediated contacts

Menisci form between two solid surfaces with the presence of an ultra-thin liquid film. Meniscus and viscous forces contribute to an adhesive force when two surfaces are separated. The adhesive force can be very large and can result in high friction, stiction and possibly high wear. The situation may become more pronounced when the contacting surfaces are ultra-smooth and the normal load is small, as is common for micro-/nanodevices. In this study, equations for meniscus and viscous forces during separation of two flat surfaces, and a sphere and a flat surface, are developed, and the corresponding adhesive forces contributed by these two types of forces are examined. The geometric meniscus curvatures and break point are theoretically determined, and the role of meniscus and viscous forces is evaluated during separation. The influence of separation distance, liquid thickness, meniscus area, separation time, liquid properties and contact angles are analyzed. Critical meniscus areas at which transition in the dominance of meniscus to viscous forces occurs for different given conditions, i.e.?various initial liquid thicknesses, contact angles and designated separation time, are identified. The analysis provides a fundamental understanding of the physics of separation process, and insights into the relationships between meniscus and viscous forces. It is also valuable for the design of the interface for various devices.     

Resolution of cross-type optical particle separation

A real-time, continuous optical particle separation method, termed cross-type optical particle separation, is investigated theoretically and experimentally. The trajectory of a particle subject to cross-type optical particle separation is predicted by solving the particle dynamic equation and compared with experimental data. For various flow velocities and particle sizes, the retention distances are measured where the displacement perpendicular to the fluid flow direction is referred to as the retention distance. The measured retention distances are in good agreement with theoretical predictions. The retention distance increases as the particle size increases due to the radiation force, but decreases as the flow velocity increases since the residence time of a particle in the laser beam decreases with increasing flow velocity. To evaluate the performance of the cross-type optical particle separation method, size-based separation resolution is derived theoretically in terms of the refractive index of the particle and instrumental fluctuations. Furthermore, an expression for the maximum resolution is derived.     

Fiber-optic microsphere-based arrays for multiplexed biological warfare agent detection

We report a multiplexed high-density DNA array capable of rapid, sensitive, and reliable identification of potential biological warfare agents. An optical fiber bundle containing 6000 individual 3.1-mum-diameter fibers was chemically etched to yield microwells and used as the substrate for the array. Eighteen different 50-mer single-stranded DNA probes were covalently attached to 3.1-mum microspheres. Probe sequences were designed for Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella melitensis, Clostridium botulinum, Vaccinia virus, and one biological warfare agent (BWA) simulant, Bacillus thuringiensis kurstaki. The microspheres were distributed into the microwells to form a randomized multiplexed high-density DNA array. A detection limit of 10 fM in a 50-microL sample volume was achieved within 30 min of hybridization for B. anthracis, Y. pestis, Vaccinia virus, and B. thuringiensis kurstaki. We used both specific responses of probes upon hybridization to complementary targets as well as response patterns of the multiplexed array to identify BWAs with high accuracy. We demonstrated the application of this multiplexed high-density DNA array for parallel identification of target BWAs in spiked sewage samples after PCR amplification. The array's miniaturized feature size, fabrication flexibility, reusability, and high reproducibility may enable this array platform to be integrated into a highly sensitive, specific, and reliable portable instrument for in situ BWA detection.     

Detection of anthrax simulants with microcalorimetric spectroscopy: Bacillus subtilis and Bacillus cereus spores

Recent advances in the development of ultrasensitive micromechnical thermal detectors have led to the advent of novel subfemtojoule microcalorimetric spectoscopy (CalSpec). On the basis of principles of photothermal IR spectroscopy combined with efficient thermomechanical transduction, CalSpec provides acquisition of vibrational spectra of microscopic samples and absorbates. We use CalSpec as a method of identifying nanogram quantities of biological micro-organisms. Our studies focus on Bacillus subtilis and Bacillus cereus spores as simulants for Bacillus anthracis spores. Using CalSpec, we measured IR spectra of B. subtilis and B. cereus spores present on surfaces in nanogram quantities (approximately 100-1,000 spores). The spectra acquired in the wavelength range of 690-4000 cm(-1) (2.5-14.5 microm) contain information-rich vibrational signatures that reflect the different ratios of biochemical makeup of the micro-organisms. The distinctive features in the spectra obtained for the two types of microorganism can be used to distinguish between the spores of the Bacillus family. As compared with conventional IR and Fourier-transform IR microscopic spectroscopy techniques, the advantages of the present technique include significantly improved sensitivity (at least a full order of magnitude), absence of expensive IR detectors, and excellent potential for miniaturization.     

Rapid chemical digestion of small acid-soluble spore proteins for analysis of Bacillus spores

A method for the rapid identification of Bacillus spores is proposed, based on the selective release and chemical digestion of small, acid-soluble spore proteins (SASPs). Microwave-assisted acid hydrolysis of SASPs from B. anthracis str. Sterne and B. subtilis str. 168 was accomplished in a single step requiring only 90 s of heating. The peptide products of the chemical digestion were identified by postsource decay sequencing with a MALDI-TOF-MS equipped with a curved-field reflectron. The specificity of the observed SASP peptides was evaluated using a cross-species sequence search. The incomplete nature of the acid digestion under these conditions allowed detection of the digest products along with the proteins from which they originated, which increased species identification confidence. The feasibility of this approach for the rapid identification of Bacillus species was further demonstrated by analyzing a mixture of B. subtilis str. 168 and B. anthracis str. Sterne spores.     

Bacillus spore classification via surface-enhanced Raman spectroscopy and principal component analysis

Surface-enhanced Raman spectroscopy (SERS) can provide rapid fingerprinting of biomaterial in a nondestructive manner. The adsorption of colloidal silver to biological material suppresses native biofluorescence while providing electromagnetic surface enhancement of the normal Raman signal. This work validates the applicability of qualitative SER spectroscopy for analysis of bacterial species by utilizing principal component analysis (PCA) to show discrimination of biological threat simulants, based upon multivariate statistical confidence limits bounding known data clusters. Gram-positive Bacillus spores (Bacillus atrophaeus, Bacillus anthracis, and Bacillus thuringiensis) are investigated along with the Gram-negative bacterium Pantoea agglomerans.     

Si芯片表面纳米粒子的粘结力及其干法清除

系统地研究了纳米粒子在硅芯片表面的各种附着力,如范德瓦耳斯力、塑性形变吸附力、毛细现象凝聚力、静电力和重力附加力,以及高速流体对它的拖动力和提拉力,并简单计算了在各种情况下这些力的大小。介绍了干法清除纳米污染物的几种新方法,如超临界流体清除法、高速气凝胶清除法、激光清除法、气相化学清除法和光化学清除法。     

Peptide-based fluorescence resonance energy transfer protease substrates for the detection and diagnosis of Bacillus species

We describe the development of a highly specific enzyme-based fluorescence resonance energy transfer (FRET) assay for easy and rapid detection both in vitro and in vivo of Bacillus spp., among which are the members of the B. cereus group. Synthetic substrates for B. anthracis proteases were designed and exposed to secreted enzymes of a broad spectrum of bacterial species. The rational design of the substrates was based on the fact that the presence of D-amino acids in the target is highly specific for bacterial proteases. The designed D-amino acids containing substrates appeared to be specific for B. anthracis but also for several other Bacillus spp. and for both vegetative cells and spores. With the use of mass spectrometry (MS), cleavage products of the substrates could be detected in sera of B. anthracis infected mice but not in healthy mice. Due to the presence of mirrored amino acids present in the substrate, the substrates showed high species specificity, and enzyme isolation and purification was redundant. The substrate wherein the D-amino acid was replaced by its L-isomer showed a loss of specificity. In conclusion, with the use of these substrates a rapid tool for detection of B. anthracis spores and diagnosis of anthrax infection is at hand. We are the first who present fluorogenic substrates for detection of bacterial proteolytic enzymes that can be directly applied in situ by the use of D-oriented amino acids.     

Optical trapping of unilamellar phospholipid vesicles: investigation of the effect of optical forces on the lipid membrane shape by confocal-Raman microscopy

Optical trapping of liposomes is a useful tool for manipulating these lipid vesicles for sampling, mechanical testing, spectroscopic observation, and chemical analysis. Through the use of confocal Raman microscopy, this study addresses the effects of optical forces on the structure of unilamellar, dipalmitoylphosphatidylcholine (DPPC) vesicles, both optically trapped in solution and adhered to a coverslip. The energy and forces involved in optical trapping of lipid vesicles were derived in terms of the dielectric contrast between the phospholipid membrane and the surrounding solution; reflection forces at the membrane/water interface were found to be negligible. At optical powers of 9 mW and greater, unilamellar liposomes trapped in bulk solution experience a gradient force sufficiently strong to bend the vesicle membrane, so that a second bilayer from the same vesicle is drawn into the optical trap, with an energy of approximately 6 x 10(-13) erg. For vesicles adhered to a coverslip, the confocal probe can be scanned through the attached vesicle. Optical forces are insufficient to detach the bilayer that is adhered to the glass; however, the upper DPPC bilayer can be manipulated by the optical trap and the shape of the vesicle distorted from a spherical geometry. The effect of calcium ion on the flexibility of membrane bilayers was also tested; with 5 mM calcium ion in solution, the lipid bilayer of a surface-attached liposome is sufficiently rigid so that it cannot be distorted at moderate laser powers.     

Development of size-selective sampling of Bacillus anthracis surrogate spores from simulated building air intake mixtures for analysis via laser-induced breakdown spectroscopy

Size-selective sampling of Bacillus anthracis surrogate spores from realistic, common aerosol mixtures was developed for analysis by laser-induced breakdown spectroscopy (LIBS). A two-stage impactor was found to be the preferential sampling technique for LIBS analysis because it was able to concentrate the spores in the mixtures while decreasing the collection of potentially interfering aerosols. Three common spore/aerosol scenarios were evaluated, diesel truck exhaust (to simulate a truck running outside of a building air intake), urban outdoor aerosol (to simulate common building air), and finally a protein aerosol (to simulate either an agent mixture (ricin/anthrax) or a contaminated anthrax sample). Two statistical methods, linear correlation and principal component analysis, were assessed for differentiation of surrogate spore spectra from other common aerosols. Criteria for determining percentages of false positives and false negatives via correlation analysis were evaluated. A single laser shot analysis of approximately 4 percent of the spores in a mixture of 0.75 m(3) urban outdoor air doped with approximately 1.1 x 10(5) spores resulted in a 0.04 proportion of false negatives. For that same sample volume of urban air without spores, the proportion of false positives was 0.08.