Continuous flow nanoparticle concentration using alternating current-electroosmotic flow.

Achieving real-time detection of environmental pathogens such as viruses and bacterial spores requires detectors with both rapid action and a suitable detection threshold. However, most biosensors have detection limits of an order of magnitude or more above the potential infection threshold, limiting their usefulness. This can be improved through the use of automated sample preparation techniques such as preconcentration. In this paper, we describe the use of AC electroosmosis to concentrate nanoparticles from a continuous flow. Electrodes at an optimized angle across a flow cell, and energized by a 1 kHz signal, were used to push nanoparticles to one side of a flow cell, and to extract the resulting stream with a high particle concentration from that side of the flow cell. A simple model of the behavior of particles in the flow cell has been developed, which shows good agreement with experimental results. The method indicates potential for higher concentration factors through cascading devices.

Node formation mechanisms in acoustofluidic capillary bridges.

Using acoustofluidic channels formed by capillary bridges two models are developed to describe nodes formed by leaky and by evanescent waves. The liquid channel held between a microscope slide (waveguide) and a strip of polystyrene film (fluid guide) avoids solid-sidewall interactions. With this simplification, our experimental and numerical study showed that waves emitted from a single plane surface, interfere and form the nodes without any resonance in the fluid. Both models pay particular attention to tensor elements normal to the solid-liquid interfaces they find that; initially nodes form in the solid and the node pattern is replicated by waves emitted into the fluid from antinodes in the stress. At fluids depths near half an acoustic wavelength, most nodes are formed by leaky waves. In the glass, water-loading reduces node-node separation and forms an overlay type waveguide which aligns the nodes predominantly along the channel. One new practical insight is that node separation can be controlled by water depth. At 0.2 mm water depths (which are smaller than a » wavelength) nodes form from evanescent waves. Here a suspension of yeast cells formed a pattern of small dot-like clumps of cells on the surface of the polystyrene film. We found the same pattern in sound intensity normal, and close, to the water-polystyrene interface. The capillary bridge channel developed for this study is simple, low-cost, and could be developed for filtration, separation, or patterning of biological species in rapid immuno-sensing applications.

Electrowetting-based Digital Microfluidics Platform for Automated Enzyme-linked Immunosorbent Assay.

Electrowetting is the effect by which the contact angle of a droplet exposed to a surface charge is modified. Electrowetting-on-dielectric (EWOD) exploits the dielectric properties of thin insulator films to enhance the charge density and hence boost the electrowetting effect. The presence of charges results in an electrically induced spreading of the droplet which permits purposeful manipulation across a hydrophobic surface. Here, we demonstrate EWOD-based protocol for sample processing and detection of four categories of antigens, using an automated surface actuation platform, via two variations of an Enzyme-Linked Immunosorbent Assay (ELISA) methods. The ELISA is performed on magnetic beads with immobilized primary antibodies which can be selected to target a specific antigen. An antibody conjugated to HRP binds to the antigen and is mixed with H2O2/Luminol for quantification of the captured pathogens. Assay completion times of between 6 and 10 min were achieved, whilst minuscule volumes of reagents were utilized.

Fully integrated digital microfluidics platform for automated immunoassay; A versatile tool for rapid, specific detection of a wide range of pathogens.

With the tangible threat posed by the release of chemical and biological warfare (CBW) agents, detection of airborne pathogens is a critical military and security concern. Recent air sampling techniques developed for biocollection take advantage of Electrowetting on Dielectric (EWOD) to recover material, producing highly concentrated droplet samples. Bespoke EWOD-based digital microfluidics platforms are very well suited to take full advantage of the microlitre concentrated droplet resulting from this recovery process. In this paper we present a free-standing, fully automated DMF platform for immunoassay. Using this system, we demonstrate the automated detection of four classes of CBW agent simulant biomolecules and organisms each representing credible threat agents. Taking advantage of the full magnetic separation process with antibody-bound microbeads, rapid and complete separation of specific target antigen can be achieved with minimal washing steps allowing for very rapid detection. Here, we report clear detection of four categories of antigens achieved with assay completion times of between six and ten minutes. Detection of HSA, Bacillus atrophaeus (BG spores), MS2 bacteriophage and Escherichia coli are demonstrated with estimated limit of detection of respectively 30 ng ml-1, 4 × 104 cfu ml-1, 106 pfu ml-1 and 2 × 107 cfu ml-1. The fully-integrated portable platform described in this paper is highly compatible with the next generation of electrowetting-coupled air samplers and thus shows strong potential toward future in-field deployable biodetection systems and could have key implication in life-changing sectors such as healthcare, environment or food security.

The application of neoglycopeptides in the development of sensitive surface plasmon resonance-based biosensors.

The development of a biosensor based on surface plasmon resonance is described for the detection of carbohydrate-binding proteins in solution on a Biacore 2000 instrument, using immobilized glycopeptides as ligands. Their selection was based on previous screenings of solid-phase glycopeptide libraries with Ricinus communis agglutinin (RCA(120)) and human adhesion/growth-regulatory galectin-1 (h-Gal-1). Glycopeptides were immobilized on Au sensor chips functionalized with mixed self-assembled monolayers of different ratios of 11-mercapto-1-undecanol and 11-mercaptoundecanoic acid, and of 3-mercapto-1-propanol and 11-mercaptoundecanoic acid. The biosensors were optimized for the detection of RCA(120), and a detection limit of 0.13 nM was obtained. Subsequent experiments with h-Gal-1 indicated a detection limit of at least 0.9 nM for this lectin. Additionally, the effect of interfering proteins on the sensitivity of the optimized biosensor was investigated.

Colorimetric detection of Ricinus communis Agglutinin 120 using optimally presented carbohydrate-stabilised gold nanoparticles.

Ricin is a toxic lectin which presents a potential security threat. Its rapid detection is highly desirable. Here we present a colorimetric bioassay based on the aggregation of carbohydrate-stabilised gold nanoparticles which has been used to detect Ricinus communis Agglutinin 120 (RCA(120)) - a ricin surrogate. To achieve a stable and robust sensing system the anchor chain length and the density of the assembled carbohydrates on the gold particle surface has been examined to determine the optimal coverage for maximal aggregation with both RCA(120) and Concanavalin A (Con A) lectins. Gold nanoparticles were stabilised with either a thiolated galactose derivative (9-mercapto-3,6-diaoxaoctyl-beta-d-galactoside) or a thiolated mannose derivative (9-merapto-3,6-dioxaoctyl-alpha-d-mannoside), for RCA(120) and Con A respectively, diluted in each instance with varying ratios of a thiolated triethylene glycol derivative. Aggregation was induced with the respective cognate lectin with the reaction monitored by UV-visible spectrophotometry. The results obtained show that a particle surface with at least 7.5% galactose is required for aggregation with RCA(120) and 6% mannose coverage is required for aggregation with Con A. For each lectin the sensitivity of the assay could be controlled by adjustment of the carbohydrate density on the gold nanoparticles, but with differing results. Maximal aggregation with Con A was achieved with a monolayer consisting of 100% mannose, whereas for RCA(120) maximal aggregation occurred with 70% coverage of galactose. The limit of detection for RCA(120) using the optimally presented galactose-stabilised nanoparticles was 9 nM.

An integrated optical leaky waveguide sensor with electrically induced concentration system for the detection of bacteria.

An integrated, sensitive and rapid system was developed for the detection of bacteria. The system combined an optical metal-clad leaky waveguide (MCLW) sensor with an electric field. The electric field was used to concentrate Bacillus subtilis var. niger(BG) bacteria spores onto the immobilized anti-BG antibody on the MCLW sensor surface. This sensor combination has been characterised by detecting the scattering from bacterial spores, which are concentrated at the sensor surface, when they are illuminated at the coupling angle; and by detection of fluorescence from labelled antibodies added after the spores had been captured on the surface. The light scattering and fluorescence detection methods gave a detection limit of BG bacterial spores of 1 x 10(3) spores ml(-1) when the electric field was applied for 3 minutes.

Bacteria detection using disposable optical leaky waveguide sensors.

Novel disposable absorbing material clad leaky waveguide sensor devices (LWD) have been developed for the detection of pathogenic particles such as bacteria. These chips are tailored to give the maximum extension of the evanescent field at the sensor surface in order to place the entire volume of the bacteria captured by immobilized antibodies on the chip surface within this field. This in turn increases the interaction of the light with the bacteria's bulk volume. Disposable LWD chips were fabricated at room temperature and without the use of expensive fabrication equipment. These LWDs have been characterised by detecting refractive index (RI) changes, scattering and fluorescence from bacterial spores at the sensor surface when illuminated at the coupling angle. The detection limit of Bacillus subtilis var. niger (BG) bacterial spores was 10(4) spores/ml and the illumination intensity of the spores was found to be three times greater than the illumination intensity generated using the surface plasmon resonance (SPR).

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.

Ultrasonic deposition of cells on a surface.

Bacteria in water have been driven to a glass surface by an ultrasonic standing wave. On an antibody coated surface capture of Bacillus subtilis var niger (BG) spores (6.6 x 10(6) ml(-1)) was increased more than 200-fold over above the efficiency in the absence of ultrasound. In microfluidic (non-turbulent) systems detection of particles by sensors operating at a surface is diffusion limited. This results in very low detection abilities particularly for particles with diameters greater than 1 microm. Ultrasound is used here to drive bacterial spores to a wall and overcome this limitation. The results confirm: (1) pressure nodes can be formed close to the water-glass interface when the glass thickness is near half the ultrasonic wavelength; (2) the antibody used was able to capture spores in the presence of an ultrasonic standing wave.

Applications of dielectrophoretic/electrohydrodynamic "zipper" electrodes for detection of biological nanoparticles.

A major problem for surface-based detection techniques such as surface plasmon resonance and quartz crystal microbalances is that at low concentrations, diffusion is an insufficient driving force to bring colloidal submicron-scale particles to the detection surface. In order to overcome this, it has previously been demonstrated that a combination of dielectrophoresis and AC-electro-hydrodynamic flow can be used to focus cell-sized particles from suspension onto a large metal surface, in order to improve the detection capabilities of such systems. In this paper we describe how the combination of these two phenomena, using the so-called "zipper" electrode array, can be used to concentrate a wide range of nanoparticles of biological interest, such as influenza virus, dissolved albumin, and DNA molecules as well as latex beads of various sizes. We also demonstrate that the speed at which particles are transported towards the centre of the electrode pads by dielectrophoresis and electro-hydrodynamic flow is not related to the particle size for colloidal particles.

Affinity determination of ricinus communis agglutinin ligands identified from combinatorial O- and S-,N-glycopeptide libraries.

Two combinatorial glycopeptide libraries were synthesized on solid support via the "split-and-mix" method combined with the ladder synthesis strategy. The O-glycopeptide library contained Gal(beta1-O)Thr, whereas the S-,N-glycopeptide library contained both Gal(beta1-S)Cys and Gal(beta1-N)Asn. In this model study, the two libraries were screened against the fluorescently labeled lectin Ricinus communis agglutinin (RCA120). The screening results showed that both O- and S- or S-,N-glycopeptides were recognized by the lectin with similar amino acid recognition patterns. Surface plasmon resonance interaction studies demonstrated that both the selected S- or S-,N-glycopeptide hits and the O-glycopeptides bound to the lectin with a similar affinity. Structure 19, containing two glycosylated cysteine residues, bound to the receptor with the highest affinity (KA = 3.81 x 10(4) M(-1)), which is comparable to N-acetyllactosamine. Competition assays, in which some selected glycopeptides and methyl beta-d-galactopyranoside competed for the binding site of immobilized RCA120, showed that the glycopeptide-lectin interaction was carbohydrate-specific. Incubation of the O- and S-,N-glycopeptides with beta-galactosidase demonstrated the complete stability of S-,N-glycopeptides toward enzymatic degradation, whereas O-glycopeptides were not completely stable.

Improvement of immunodetection of bacterial spore antigen by ultrasonic cavitation.

Ultrasonic cavitation was employed to enhance sensitivity of bacterial spore immunoassay detection, specifically, enzyme-linked immunosorbent assay (ELISA) and resonant mirror (RM) sensing. Bacillus spore suspensions were exposed to high-power ultrasound in a tubular sonicator operated at 267 kHz in both batch and flow modes. The sonicator was designed to deliver high output power and is in a form that can be cooled efficiently to avoid thermal denaturation of antigen. The 30-s batch and cooled flow (0.3 mL/min) sonication achieved an approximately 20-fold increase in ELISA sensitivity compared to unsonicated spores by ELISA. RM sensing of sonicated spores achieved detection sensitivity of approximately 10(6) spores/mL, whereas unsonicated spores were undetectable at the highest concentration tested. Improvements in detection were associated with antigen released from the spores. Equilibrium temperature increase in the tubular sonicator was limited to 14 K after 30 min and was maintained for 6 h with cooling and flow (0.3 mL/min). The work described here demonstrates the utility of the tubular sonicator for the improvement in the sensitivity of the detection of spores and its suitability as an in-line component of a rapid detection system.

An integrated metal clad leaky waveguide sensor for detection of bacteria.

An integrated optical metal clad leaky waveguide (MCLW) sensor device has been developed for the detection of bacteria. This is more sensitive than waveguide sensors currently in use. The MCLW device has been fabricated to extend the evanescent field to provide significant light intensity over the entire volume of the bacteria bound on the chip surface within this field. This in turn increases the interaction of the light with the entire volume of the bacteria. MCLW devices have been used for detecting refractive index changes, scattering, and fluorescence from bacterial spores captured on an immobilized antibody. The detection limit of Bacillus subtilis var. niger bacterial spores using refractive index detection was 8 x10(4) spores/mL. The scattering intensity of the BG spores was found to be three times greater than the scattering intensity generated using surface plasmon resonance. The extended light propagation along the direction of flow for a few millimeters provides an effective interrogation approach to increase the area of detection to detect low concentrations down to 1 x 10(4) spores/mL. The sensor was then optimized by studying the key factors affecting sensor performance including changing the pH of the medium, type of antibody immobilization matrix, sensor surface regeneration approaches, and longevity of the sensor.

Optical leaky waveguide sensor for detection of bacteria with ultrasound attractor force.

An integrated, sensitive, and rapid system was developed for the detection of bacteria. The system combined an optical metal-clad leaky waveguide (MCLW) sensor with ultrasound standing waves (USW). The performance of a MCLW sensor for the detection of bacteria has been increased (>100 fold) by using USWs to drive bacteria onto the sensor surface. By forming the USW nodes at or within the surface of the MCLW, the diffusion-limited capture rate has been replaced by fast movement. Immobilized anti-BG antibody on the MCLW sensor surface was used to capture Bacillus subtilis var. niger (BG) bacterial spores driven to the surface. This combination of sensor and attractor force combination has been tested by detecting the evanescent scattering from bacterial spores at the sensor surface. Application of ultrasound for 3 min gave a detection limit for BG bacterial spores of 1 x 10(3) spores/mL.