Spore and micro-particle capture on an immunosensor surface in an ultrasound standing wave system

The capture of Bacillus subtilis var. niger spores on an antibody-coated surface can be enhanced when that coated surface acts as an acoustic reflector in a quarter wavelength ultrasonic (3 MHz) standing wave resonator. Immunocapture in such a resonator has been characterised here for both spores and 1 microm diameter biotinylated fluorescent microparticles. A mean spatial acoustic pressure amplitude of 460 kPa and a frequency of 2.82 MHz gave high capture efficiencies. It was shown that capture was critically dependent on reflector thickness. The time dependence of particle deposition on a reflector in a batch system was broadly consistent with a calculated time of 35 s to bring 95% of particles to the coated surface. A suspension flow rate of 0.1 ml/min and a reflector thickness of 1.01 mm gave optimal capture in a 2 min assay. The enhancement of particle detection compared with the control (no ultrasound) situation was x 70. The system detects a total of five particles in 15 fields of view in a 2 min assay when the suspending phase concentration was 10(4) particles/ml. A general expression for the dependence of minimum concentration detectable on; number of fields examined, sample volume flowing through the chamber and assay time shows that, for a practical combination of these variables, the threshold detection concentration can be two orders of magnitude lower.     

Applications of ultrasound streaming and radiation force in biosensors

Direct radiation force (DRF) and acoustic streaming provide the main influences on the behaviour of particles in aqueous suspension in an ultrasound standing wave (USW). The direct radiation force, which drives suspended particles towards and concentrates them in acoustic pressure node planes, has been applied to rapidly transfer cells in small scale analytical separators. The DRF also significantly increased the sensitivity of latex agglutination test (LAT) by concentrating the particles of an analytical sample in the pressure node positions and hence greatly increasing the antibody-antigen encounter rate. Capture of biotinylated particles and spores on a coated acoustic reflector in a quarter wavelength USW resonator was DRF-enhanced by 70- and 100-fold, respectively compared to the situation in the absence of ultrasound. Acoustic streaming has been successfully employed for mixing small analytical samples. Cavitation micro-streaming substantially enhanced, through mixing, DNA hybridization and the capture efficiency of Escherichia coli K12 on the surface of immunomagnetic beads. Acoustic streaming induced in longitudinal standing wave and flexural plate wave immuno-sensors increased the detection of antigens by a factor of five and three times, respectively. Combined DRF and acoustic streaming effects enhanced the rate of the reaction between suspended mixture of cells and retroviruses. The examples of a biochip and an ultrasonic immuno-sensor implementing the DRF and acoustic streaming effects are also described in the review.     

Development of instrumentation to allow the detection of microorganisms using light scattering in combination with surface plasmon resonance

This paper describes work carried out to develop a biosensor which allows two separate detection principles to operate simultaneously at the same surface. A prototype device was constructed that provided Kretschmann-configuration surface plasmon resonance (SPR) measurement of refractive index (RI) changes using an 820 nm LED light source, whilst a 635 nm diode laser was used to produce light scattering signals from bacterial spores. Both effects occurred at a gold-coated surface. The RI changes were measured conventionally from the side of the gold layer nearer to the light sources. The scattered light was imaged from the opposite face which was in contact with the aqueous sample. Specific detection of bacterial spores through the light scattering mode using antibody capture was investigated. The flow dynamics and interactions with the surface of individual spores were observed. A comparison with SPR for detection using the same antibody/antigen pair was made. Spore suspensions that were readily detectable by light scattering at 10(7) ml(-1) did not provide significant responses by SPR. The potential for future developments is discussed.     

Low-level detection of a bacillus anthracis simulant using Love-wave biosensors on 36 degrees YX LiTaO3

We present an acoustic Love-wave biosensor for detection of the Bacillus anthracis simulant, Bacillus thuringiensis at or below inhalational infectious levels. The present work is an experimental study of 36 degrees YX cut LiTaO3 based Love-wave devices for detection of pathogenic spores in aqueous conditions. Given that the detection limit (D1) of Love-wave-based sensors is a strong function of the overlying waveguide, two waveguide materials have been investigated, which are polyimide and polystyrene. To determine the mass sensitivity of Love-wave sensor, bovine serum albumin (BSA) protein was injected into the Love-wave test cell while recording the magnitude and phase shift across each sensor. Polyimide had the lowest mass detection limit with an estimated value of 1.0-2.0 ng/cm2, as compared to polystyrene where D1 = 2.0 ng/cm2. Suitable chemistries were used to orient antibodies on the Love-wave sensor using protein G. The thickness of each biofilm was measured using ellipsometry from which the surface concentrations were calculated. The monoclonal antibody BD8 with a high degree of selectivity for anthrax spores was used to capture the non-pathogenic simulant B. thuringiensis B8 spores. Bacillus subtilis spores were used as a negative control to determine whether significant non-specific binding would occur. Spore aliquots were prepared using an optical counting method, which permitted removal of background particles for consistent sample preparation. This work demonstrates that Love-wave biosensors are promising for low-level detection for whole-cell biological pathogens.     

Sub-micron particle manipulation in an ultrasonic standing wave: Applications in detection of clinically important biomolecules

Separation of particles from the suspending phase is of interest, among others, to clinical analysts. A system that enables manipulation of sub-micron sized particles in suspensions of analytical scale volume (10–50 l) using a non-cavitating ultrasonic standing wave is described. Particle suspensions, contained in glass capillary tubes of 1–2 mm internal dimension, are treated on the axis of a tubular transducer generating a radial standing wave field at 4.5 MHz. Microparticles (of average diameter range 0.3–10 m) suspended in buffer are concentrated within seconds at preferred regions separated by submillimetre distances. Concentration of suspended latex particles was inhibited in solutions containing protein at levels similar to those occurring in clinical specimens when the suspensions were sonicated in capillaries of circular cross-section. This effect was associated with acoustic streaming of the suspending fluid. Silica microparticles (more dense and less compressible than latex) could be concentrated in the presence of streaming. Latex particles concentrated readily in square cross-section capillaries where no streaming was observed. With sub-micron particles, the geometry of the sample chamber, the suspending phase composition and the size, density and compressibility of the microparticles all influence particle manipulation. The radial standing wave system has been used to enhance agglutination of antibody-coated latex microparticles in the presence of antigen allowing rapid and highly sensitive detection of clinically important biomolecules. The sensitivity of conventional diagnostic tests for microbial antigen has been improved by application of ultrasound and clinical utility has been demonstrated, in particular, for detection of meningitis-causing bacteria.     

Three-Dimensional Mid-Air Acoustic Manipulation by Ultrasonic Phased Arrays

The essence of levitation technology is the countervailing of gravity. It is known that an ultrasound standing wave is capable of suspending small particles at its sound pressure nodes. The acoustic axis of the ultrasound beam in conventional studies was parallel to the gravitational force, and the levitated objects were manipulated along the fixed axis (i.e. one-dimensionally) by controlling the phases or frequencies of bolted Langevin-type transducers. In the present study, we considered extended acoustic manipulation whereby millimetre-sized particles were levitated and moved three-dimensionally by localised ultrasonic standing waves, which were generated by ultrasonic phased arrays. Our manipulation system has two original features. One is the direction of the ultrasound beam, which is arbitrary because the force acting toward its centre is also utilised. The other is the manipulation principle by which a localised standing wave is generated at an arbitrary position and moved three-dimensionally by opposed and ultrasonic phased arrays. We experimentally confirmed that expanded-polystyrene particles of 0.6 mm, 1 mm, and 2 mm in diameter could be manipulated by our proposed method.     

The measurement of Bacillus mycoides spore adhesion using atomic force microscopy,simple counting methods,and a spinning disk technique

An atomic force microscope has been used to study the adhesion of Bacillus mycoides spores to a hydrophilic glass surface and a hydrophobic-coated glass surface. AFM images of spores attached to the hydrophobic-coated mica surface allowed the measurement of spore dimensions in an aqueous environment without desiccation. The spore exosporium was observed to be flexible and to promote the adhesion of the spore by increasing the area of spore contact with the surface. Results from counting procedures using light microscopy matched the density of spores observed on the hydrophobic-coated glass surface with AFM. However, no spores were observed on the hydrophilic glass surface with AFM, a consequence of the weaker adhesion of the spores at this surface. AFM was also used to quantify directly the interactions of B. mycoides spores at the two surfaces in an aqueous environment. The measurements used "spore probes" constructed by immobilizing a single spore at the apex of a tipless AFM cantilever. The data showed that stretching and sequential bond breaking occurred as the spores were retracted from the hydrophilic glass surface. The greatest spore adhesion was measured at the hydrophobic-coated glass surface. An attractive force on the spores was measured as the spores approached the hydrophobic-coated surface. At the hydrophilic glass surface, only repulsive forces were measured during the approach of the spores. The AFM force measurements were in qualitative agreement with the results of a hydrodynamic shear adhesion assay that used a spinning disk technique. Quantitatively, AFM measurements of adhesive force were up to 4 x 10(3) times larger than the estimates made using the spinning disk data. This is a consequence of the different types of forces applied to the spore in the different adhesion assays. AFM has provided some unique insights into the interactions of spores with surfaces. No other instrument can make such direct measurements for single microbiological cells.     

Immuno-capture of Cryptosporidium parvum using micro-well array

A glass slide and micro-well array chip on which anti-Cryptosporidium parvum antibody was immobilized were used for the rapid capture and detection of C. parvum. Biotinylated anti-C. parvum antibodies were spotted onto the streptavidin-coated glass slides. C. parvum oocysts were captured specifically on the spot when more than 73 ng of anti-C. parvum antibody was applied onto the glass slide. However, C. parvum oocysts captured on the glass slide were detached by repeating washing steps. To improve the capture efficiency of oocysts, capture was performed in a micro-well format consisting of 1024 wells/2.5 mm2 (32 x 32 wells) fabricated as a chip by photolithography. Instead of a flat surface on a glass slide, each well was 30 microm in diameter and 10 microm in depth. Streptavidin was also immobilized onto the micro-well array. The biotinylated anti-C. parvum antibodies were immobilized efficiently onto the chip using a buffer containing 20% methanol. Using this technique C. parvum oocysts were stably captured onto the chip after repeated washing procedures. These data show that the newly designed micro-well array technique described here is useful for antibody-mediated C. parvum capture.     

Sub-micron particle behaviour and capture at an immuno-sensor surface in an ultrasonic standing wave

The capture of 200 nm biotinylated latex beads from suspensions of concentration 10(7) to 2.5 x 10(8) particle/ml on an immuno-coated surface of the acoustic reflector in an ultrasound standing wave (USW) resonator has been studied while the acoustic pathlength was less than one half wavelength (lambda/2). The particles were delivered to the reflector's surface by acoustically induced flow. The capture dependencies on suspension concentration, duration of experiments and acoustic pressure have been established at 1.09, 1.46 and 1.75 MHz. Five-fold capture increase has been obtained at 1.75 MHz in comparison to the control (no ultrasound) situation. The contrasting behaviours of 1, 0.5 and 0.2 mum fluorescent latex beads in a lambda/4 USW resonator at 1.46 MHz have been characterized. The particle movements were observed with an epi-fluorescent microscope and the velocities of the particles were measured by particle image velocimetry (PIV). The experiments showed that whereas the trajectories of 1 mum particles were mainly affected by the direct radiation force, 0.5 mum particles were influenced both by the radiation force and acoustic streaming. The 0.2 mum latex beads followed acoustic streaming in the chamber and were not detectably affected by the radiation force. The streaming-associated behaviour of the 200 nm particles has implications for enhanced immunocapture of viruses and macromolecules (both of which are also too small to experience significant acoustic radiation force).     

Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor

Since the anthrax spore bioterrorism attacks in America in 2001, the early detection of Bacillus anthracis spores and vegetative cells has gained significant interest. At present, many polyclonal antibody-based quartz crystal microbalance (QCM) sensors have been developed to detect B. anthracis simulates. To achieve a simultaneous rapid detection of B. anthracis spores and vegetative cells, this paper presents a biosensor that utilizes an anti-B. anthracis monoclonal antibody designated to 8G3 (mAb 8G3, IgG) functionalized QCM sensor. Having compared four kinds of antibody immobilizations on Au surface, an optimized mAb 8G3 was immobilized onto the Au electrode with protein A on a mixed self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) and 6-mercaptohexan-1-ol (6-MHO) as adhesive layer. The detection of B. anthracis was investigated under three conditions: dip-and-dry, static addition and flow through procedure. The results indicated that the sensor yielded a distinct response to B. anthracis spores or vegetative cells but had no significant response to Bacillus thuringiensis species. The functionalized sensor recognized B. anthracis spores and vegetative cells specifically from its homophylic ones, and the limit of detection (LOD) reached 10(3)CFU or spores/ml of B. anthracis in less than 30 min. Cyclic voltammogram (CV) and scanning electronic microscopy (SEM) were performed to characterize the surface of the sensor in variable steps during the modification and after the detection. The mAb functionalized QCM biosensor will be helpful in the fabrication of a similar biosensor that may be available in anti-bioterrorism in the future.     

Solid-phase capture of proteins, spores, and bacteria

Current methods for the detection of pathogens in food and water samples generally require a preenrichment step that allows selective enrichment of the test organism. The objective of this research was to eliminate an enrichment step to allow detection of bacteria directly in food and water samples in 30 min. A high-flow-rate, fluidized bed to capture and concentrate large (bacteria and spores) and small (protein) molecules was developed. This format, ImmunoFlow, is volume independent and uses large beads (greater than 3 mm in diameter) when capturing bacteria to prevent sample clogging when testing food samples. Detection of bound targets was done using existing enzyme-linked immunosorbent assay (ELISA) protocols. Four antibodies (anti-Escherichia coli O157:H7, -Bacillus globigii, -bovine serum albumin [BSA], and -ovalbumin [OVA]) were covalently coupled to various glass and ceramic beads. Very small amounts of BSA (<1 ng) and OVA (0.2 to 4.0 microg) were detected. Various industrial and environmental samples were used to observe the effect of the sample composition on the capture of anti-B. globigii and anti-E. coli O157:H7 modified beads. The lower limit of detection for both E. coli O157:H7 and B. globigii was 1 spore/cell independent of the sample size. The activity of anti-B. globigii modified beads declined after 3 days. Anti-E. coli O157:H7 modified beads declined in their capture ability after 2 days in various storage buffers. Storage temperature (4 and 25 degrees C) did not influence the stability. The ImmunoFlow technology is capable of capturing bacteria and spores directly from samples, with subsequent detection in an ELISA format in 30 min.     

Dynamics of rebounding <Emphasis Type="Italic">Bacillus subtilis</Emphasis> spores determined using image-charge detection

A novel image-charge detection technique was used to investigate the mechanical elasticity of bare bacterial spores during high-velocity impact. Spores of Bacillus subtilis introduced to vacuum using electrospray and aerodynamic acceleration impacted and rebounded off of a glass plate. A dual-stage, asymmetric image-charge detector measured the velocity and direction of each spore both before and after impact with the glass surface. Two ranges of impact velocity were investigated, with average initial velocities of 197 ± 17 and 145 ± 12 m/s. Impacts were strongly inelastic, with most of the translational kinetic energy lost upon impact, similar to polystyrene particles of similar size under similar impact velocities. Specifically, 69% (± 16%) and 74% (± 11%) of initial kinetic energy was lost in impacts at the two velocity ranges, respectively. The average coefficients of restitution for the two velocity regimes were 0.53 ± 0.15 and 0.49 ± 0.12. There was no statistically significant difference in the fractional kinetic energy loss between these two populations. The variance of these results is much larger than experiments using polystyrene spheres of comparable size. These results imply significant plastic deformation of the spore—a striking result given that spores of this strain of B. subtilis are known to survive impacts on glass at these velocities. Triboelectric charge transfer during impact was also observed. Although much is known about spore elasticity from static measurements, this is the first study to investigate the elastic properties of bacterial spores in a dynamic scenario, as well as the first demonstration of an image charge detector for measurements of rebounding particles.     

Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces

We present a generic immunoassay platform that uses enhanced total internal reflection fluorescence in the proximity of silver island films (SIFs), a surface coating consisting of metal (silver) particles. This platform is used with a model immunoassay where a protein antigen, rabbit immunoglobulin G, was immobilized on the SIF-coated glass surface. The signal from a fluorescent dye-labeled anti-rabbit antibody binding to the surface antigen was detected; different color dyes have been tested. Close placement of the fluorophore to surface-bound silver nanostructures results in dramatic signal enhancement (up to 40-fold) on the SIFs as compared with the glass slides. Use of the total internal reflection mode of excitation has significant advantages (over classic front-face excitation) for practical assay development. The limited evanescent wave excitation volume makes it possible to minimize the background signal and use the immunoassay with no need for any washing steps.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Use of ultrasonic energy in assessing microbial contamination on surfaces

Ultrasonic tanks were evaluated for their ability to remove viable microorganisms from various surfaces for subsequent enumeration. Test surfaces were polished stainless steel, smooth glass, frosted glass, and electronic components. The position of contaminated surfaces in relation to the ultrasonic energy source, distance of the ultrasonic source from the test surfaces, and temperature of the rinse fluid were some of the factors which influenced recovery. Experimental systems included both naturally occurring microbial contamination and artificial contamination with spores of Bacillus subtilis var. niger. The results showed that ultrasonic energy was more reliable and efficient than mechanical agitation for recovering surface contaminants. Conditions which increased the number and percentage of microorganisms recovered by ultrasonic energy were: using a cold rinse fluid, placing the sample bottle on the bottom of the ultrasonic tank, and facing the contaminated surfaces toward the energy source. It was also demonstrated that ultrasonic energy could be effectively used for eluting microorganisms from cotton swabs.     

Gold nanoparticle-based detection of genomic DNA targets on microarrays using a novel optical detection system

The development of a nanoparticle-based detection methodology for sensitive and specific DNA-based diagnostic applications is described. The technology utilizes gold nanoparticles derivatized with thiol modified oligonucleotides that are designed to bind complementary DNA targets. A glass surface with arrays of immobilized oligonucleotide capture sequences is used to capture DNA targets, which are then detected via hybridization to the gold nanoparticle probes. Amplification with silver allows for detection and quantitation by measuring evanescent wave induced light scatter with low-cost optical detection systems. Compared to Cy3-based fluorescence, silver amplified gold nanoparticle probes provide for a approximately 1000-fold increase in sensitivity. Furthermore, direct detection of non-amplified genomic DNA from infectious agents is afforded through increased specificity and even identification of single nucleotide polymorphisms (SNP) in human genomic DNA appears feasible.     

Comparative surface examinations of morse taper junctions of the MRP-Titan stem shot peened with glass beads]

INTRODUCTION: Shot peening is widely used for surface treatment of hip implants. Shot peening with steel balls followed by a cleaning process with glass beads is used for introduction of negative stress in the production of morse taper junctions of the MRP-Titan stem. An increasing number of publications in maxillofacial surgery and orthopaedic surgery show that there is a significant contamination of Alumina or glass blasted surfaces. Latest research suggested an association between contaminant particles with early loosening of endoprostheses (third body wear). The aim of this study is to evaluate the amount and the effects of surface contamination with glass particles on morse taper junctions of implants and explants of the MRP-Titan stem. MATERIAL AND METHOD: The surface of morse taper junctions of the MRP-Titan stem (5 original-package implants and explants each) are analysed for glass particle contamination. A field emission scanning electron microscopy (LEO 1525) is used for the detection of the glass-particles on the implant surface with a backscattered electron detector. The relative surface area covered by particles was calculated by means of an image analyzing software (analySIS, Soft Imaging System GmbH). RESULTS: The surface of the implants showed a considerable contamination with glass particles with a mean of 6.67 +/- 0.82% compared to 2.06 +/- 0.74% on the surface of the explants. The difference was statistically significant (p<0.0001). DISCUSSION: The results of this study show that there is a relative high percentage of contamination with glass particles on shot peened morse taper junctions of the MRP-Titan stem. This contamination is significantly lower on the surface of the explants. With respect to third body wear and osteolysis in total hip arthroplasty further studies are necessary to minimize contamination while maintaining adequate surface quality.     

Use of a Foam Spatula for Sampling Surfaces after Bioaerosol Deposition

The present study had three goals: (i) to evaluate the relative quantities of aerosolized Bacillus atrophaeus spores deposited on the vertical, horizontal top, and horizontal bottom surfaces in a chamber; (ii) to assess the relative recoveries of the aerosolized spores from glass and stainless steel surfaces with a polyester swab and a macrofoam sponge wipe; and (iii) to estimate the relative recovery efficiencies of aerosolized B. atrophaeus spores and Pantoea agglomerans using a foam spatula at several different bacterial loads by aerosol distribution on glass surfaces. The majority of spores were collected from the bottom horizontal surface regardless of which swab type and extraction protocol were used. Swabbing with a macrofoam sponge wipe was more efficient in recovering spores from surfaces contaminated with high bioaerosol concentrations than swabbing with a polyester swab. B. atrophaeus spores and P. agglomerans culturable cells were detected on glass surfaces using foam spatulas when the theoretical surface bacterial loads were 2.88 × 10⁴ CFU and 8.09 × 10⁶ CFU per 100-cm² area, respectively. The median recovery efficiency from the surfaces using foam spatulas was equal to 9.9% for B. atrophaeus spores when the recovery was calculated relative to the theoretical surface spore load. Using a foam spatula permits reliable sampling of spores on the bioaerosol-exposed surfaces in a wide measuring range. The culturable P. agglomerans cells were recovered with a median efficiency of 0.001%, but staining the swab extracts with fluorescent dyes allowed us to observe that the viable cell numbers were higher by 1.83 log units than culturable organisms. However, additional work is needed to improve the analysis of the foam extracts in order to decrease the limit of detection of Bacillus spores and Gram-negative bacteria on contaminated surfaces.Surface sampling is performed on a frequent basis in all situations where clean environment monitoring is needed, e.g., in health care facilities and in the pharmaceutical industry and food industry. An anthrax bioterrorist event in the fall of 2001 has emphasized the importance of efficient sampling methods for detection of pathogenic microorganisms on surfaces within intentionally contaminated locations (22). Unfortunately, our knowledge on the most effective sampling methodology as well as the level of confidence we may have in the results obtained by wiping, swabbing, and other sample collection strategies is still limited (1). Moreover, in most of the studies performed so far, bacteria and/or spores were collected from test samples or coupons of various materials, inoculated with a suspension of microorganisms that had been placed and spread over the surface, and then dried (14, 15). This may not mimic the true situation of surface contamination by a pathogen that has been intentionally released. Edmonds et al. (12) recently reported lower swabbing efficiencies of different types of swab materials used for sampling glass, polycarbonate, and vinyl surfaces contaminated with dry aerosol-deposited Bacillus atrophaeus spores compared to the surfaces inoculated by spore suspensions. Solid surface contamination from exposure to aerosolized spores fits the real world better than the previous models.Therefore, in our study we decided to generate aerosols of various concentrations of B. atrophaeus spores as well as the vegetative cells of Pantoea agglomerans inside a chamber where the bioaerosol particles were allowed to gravitationally settle on solid surfaces. The aerosolization of P. agglomerans was performed to verify the recovery of Gram-negative bacteria according to the recommendations of Budowle et al. (5). The main goal of our study was to establish the range of detection when bioaerosol-contaminated surfaces were swabbed using a commercially available foam spatula.     

Magnetic scanometric DNA microarray detection of methyl tertiary butyl ether degrading bacteria for environmental monitoring

A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 μm diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 μm diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications.     

Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications

We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits.