High sensitivity detection of molecular recognition using magnetically labelled biomolecules and magnetoresistive sensors

Small magnetoresistive spin valve sensors (2 x 6 microm(2)) were used to detect the binding of single streptavidin functionalized 2 microm magnetic microspheres to a biotinylated sensor surface. The sensor signals, using 8 mA sense current, were in the order of 150-400 microV for a single microsphere depending on sensor sensitivity and the thickness of the passivation layer over the sensor surface. Sensor saturation signals were 1-2 mV representing an estimated 6-20 microspheres, with a noise level of approximately 10 microV. The detection of biomolecular recognition for the streptavidin-biotin model was shown using both single and differential sensor architectures. The signal data compares favourably with previously reported signals for high numbers of magnetic microspheres detected using larger multilayered giant magnetoresistance sensors. A wide range of applications is foreseen for this system in the development of biochips, high sensitivity biosensors and the detection of single molecules and single molecule interactions.     

Direct protein detection with a nano-interdigitated array gate MOSFET

A new protein sensor is demonstrated by replacing the gate of a metal oxide semiconductor field effect transistor (MOSFET) with a nano-interdigitated array (nIDA). The sensor is able to detect the binding reaction of a typical antibody Ixodes ricinus immunosuppressor (anti-Iris) protein at a concentration lower than 1 ng/ml. The sensor exhibits a high selectivity and reproducible specific detection. We provide a simple model that describes the behavior of the sensor and explains the origin of its high sensitivity. The simulated and experimental results indicate that the drain current of nIDA-gate MOSFET sensor is significantly increased with the successive binding of the thiol layer, Iris and anti-Iris protein layers. It is found that the sensor detection limit can be improved by well optimizing the geometrical parameters of nIDA-gate MOSFET. This nanobiosensor, with real-time and label-free capabilities, can easily be used for the detection of other proteins, DNA, virus and cancer markers. Moreover, an on-chip associated electronics nearby the sensor can be integrated since its fabrication is compatible with complementary metal oxide semiconductor (CMOS) technology.     

Magnetoresistive-based real-time cell phagocytosis monitoring

The uptake of large particles by cells (phagocytosis) is an important factor in cell biology and also plays a major role in biomedical applications. So far, most methods for determining the phagocytic properties rely on cell-culture incubation and end-point detection schemes. Here, we present a lab-on-a-chip system for real-time monitoring of magnetic particle uptake by human fibroblast (NHDF) cells. It is based on recording the time evolution of the average position and distribution of magnetic particles during phagocytosis by giant-magnetoresistive (GMR) type sensors. We employ particles with a mean diameter of 1.2 μm and characterize their phagocytosis-relevant properties. Our experiments at physiological conditions reveal a cellular uptake rate of 45 particles per hour and show that phagocytosis reaches saturation after an average uptake time of 27.7h. Moreover, reference phagocytosis experiments at 4°C are carried out to mimic environmental or disease related inhibition of the phagocytic behavior, and our measurements clearly show that we are able to distinguish between cell-membrane adherent and phagocytosed magnetic particles. Besides the demonstrated real-time monitoring of phagocytosis mechanisms, additional nano-biointerface studies can be realized, including on-chip cell adhesion/spreading as well as cell migration, attachment and detachment dynamics. This versatility shows the potential of our approach for providing a multifunctional platform for on-chip cell analysis.     

Nanostructured digital microfluidics for enhanced surface plasmon resonance imaging

The advances in genomics and proteomics have unveiled an exhaustive catalogue of biomarkers that can potentially be used as diagnostic and prognostic indicators of genetic and infectious diseases. Current thrust in biosensor development is towards rapid, real-time, label-free and highly sensitive detection of the indicative biomarkers. While surface plasmon resonance imaging (SPRi) biosensors could potentially be the best suited candidate for biomarker-based diagnosis, important milestones need to be reached. Commercially available SPRi instrumentation is currently limited by the flow-cell technology to serial-sample processing and has limited sensitivity for the detection of markers present at low concentration. In this paper, we have implemented an approach to enhance sample handling and increase the sensitivity of the SPRi detection technique. We have developed a digital microfluidic platform with an integrated nanostructured biosensor interface that allows for rapid, ultra-low volume, sensitive, and automated on-chip SPRi detection of DNA hybridization reactions. Through the exploitation of electromagnetic properties of nanofabricated periodic gold nanoposts, SPRi signal was increased by 200% with the estimated limit of detection of 500 pM (90 attomoles). Using the versatile fluidic manipulation provided by the digital microfluidics, rapid and parallel target identification was achieved on multiple array elements within 1 min using 180 nL sample volume. By delivering multiple target analytes in individually addressable low volume droplets, without external pumps and fluidic interconnects, the overall assay time, cost and complexity was reduced. The proposed platform allows extreme versatility in the manipulation of precious low volume samples which makes this technology very suitable for diagnostic applications.     

Analysing a magnetic molecule detection system--computer simulation

The detection of single molecules, e.g. in biology is possible by marking the interesting molecules with magnetic beads and detect the influence of the beads on giant magnetoresistance (GMR)/tunnel magnetoresistance (TMR)/spin valve (SV) sensors. The development of suitable multilayers has been studied experimentally as well as theoretically in order to optimize the sensor parameters. A finite difference (FD) method including the usually used contributions to the total energy [exchange, antiferromagnetically (af) coupling, anisotropy and magnetostatic] is used for the simulation with additional contributions to the local field according to the stray fields of the beads. In this work, we will show the results of micromagnetic calculations of the magnetization behavior of GMR/TMR sensors considering also the interaction between the domains in the magnetic layers of the sensor and the bead area. We can present first calculations where the bead particles (signal source) and the magnetic layers (sensor device) are considered as a whole magnetic ensemble.     

Application of surface plasmon resonance imaging technique for the detection of single spherical biological submicrometer particles

Recent proof-of-principle studies demonstrated the suitability of the surface plasmon resonance imaging (SPRi) technique for the detection of individual submicrometer and nanoparticles in solutions. In the current study, we used the SPRi technique for visualization of the binding of round-shaped viruses (inactivated influenza A virus) and virus-like particles (human immunodeficiency virus (HIV)-based virus-like particles) to the functionalized sensor surface. We show the applicability of the SPRi technique for the detection of individual virus-like particles in buffers without serum as well as in buffers containing different concentrations of serum. Furthermore, we prove the specificity of visualized binding events using two different pseudotypes of HIV virus-like particles. We also demonstrate the applicability of the SPRi technique for the determination of relative particle concentrations in solutions. Moreover, we suggest a technical approach, which allows enhancing the magnitude of binding signals. Our studies indicate that the SPRi technique represents an efficient research tool for quantification and characterization of biological submicrometer objects such as viruses or virus-like particles, for example.     

Monitoring the growth and drug susceptibility of individual bacteria using asynchronous magnetic bead rotation sensors

Continuous growth of individual bacteria has been previously studied by direct observation using optical imaging. However, optical microscopy studies are inherently diffraction limited and limited in the number of individual cells that can be continuously monitored. Here we report on the use of the asynchronous magnetic bead rotation (AMBR) sensor, which is not diffraction limited. The AMBR sensor allows for the measurement of nanoscale growth dynamics of individual bacterial cells, over multiple generations. This torque-based magnetic bead sensor monitors variations in drag caused by the attachment and growth of a single bacterial cell. In this manner, we observed the growth and division of individual Escherichia coli, with 80-nm sensitivity to the cell length. Over the life cycle of a cell, we observed up to a 300% increase in the rotational period of the biosensor due to increased cell volume. In addition, we observed single bacterial cell growth response to antibiotics. This work demonstrates the non-microscopy limited AMBR biosensor for monitoring individual cell growth dynamics, including cell elongation, generation time, lag time, and division, as well as their sensitivity to antibiotics.     

Solubilization and display of G protein-coupled receptors on beads for real-time fluorescence and flow cytometric analysis

G protein-coupled receptors (GPCR) and cellular signaling elements are prime targets for drug discovery. Sensitive real-time methods that expand the analytical capabilities for these elements can play significant roles in basic research and drug discovery. Here, we describe novel approaches for the real-time fluorescence analysis of GPCRs. Using the G protein-coupled N-formyl peptide receptor (FPR) as a model system in concert with a fluorescent ligand, we showed the quantitative solubilization of his-tagged FPRs in 1% dodecyl maltoside. Solubilized receptors reconstitute in dodecyl maltoside with a mixture of bovine brain Gi/Go showing an apparent Kd of 100 nM. Solubilized receptors were also bound to Ni(2+)-silica particles and were detected in a flow cytometer by the binding of fluorescent ligand. The efficiency of receptor uptake by the particles was in excess of 80% with an apparent affinity for the bead in the nM range. The receptors had largely homogeneous dissociation characteristics, an appropriate Kd for the ligand in the low nM range and a high site number, with several million receptor molecules per particle. However, the G protein reconstitution was not detected on the beads, apparently for steric reasons. These approaches for displaying receptors could prove useful in drug discovery and in the analysis of the molecular assemblies in signal transduction.     

Dual signal amplification of surface plasmon resonance imaging for sensitive immunoassay of tumor marker

Surface plasmon resonance imaging (SPRi) is an intriguing technique for immunoassay with the inherent advantages of being high throughput, real time, and label free, but its sensitivity needs essential improvement for practical applications. Here, we report a dual signal amplification strategy using functional gold nanoparticles (AuNPs) followed by on-chip atom transfer radical polymerization (ATRP) for sensitive SPRi immunoassay of tumor biomarker in human serum. The AuNPs are grafted with an initiator of ATRP as well as a recognition antibody, where the antibody directs the specific binding of functional AuNPs onto the SPRi sensing surface to form immunocomplexes for first signal amplification and the initiator allows for on-chip ATRP of 2-hydroxyethyl methacrylate (HEMA) from the AuNPs to further enhance the SPRi signal. High sensitivity and broad dynamic range are achieved with this dual signal amplification strategy for detection of a model tumor marker, α-fetoprotein (AFP), in 10% human serum.     

DNA hybridization detection with organic thin film transistors: toward fast and disposable DNA microarray chips

We demonstrate a novel DNA hybridization detection method with organic thin film transistors. DNA molecules are immobilized directly on the surface of organic semiconductors, producing an unambiguous doping-induced threshold voltage shift upon hybridization. With these shifts, single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) are differentiated successfully. This method is expected to result in higher sensitivity than the main competitive technology, ISFET-based sensors because of the direct exposure of DNA molecules to sensitive layers. Factors that influence sensor sensitivity have been analyzed and optimum conditions have been determined using statistically designed experiments. Under the optimum conditions, the maximum difference between saturation current ratios caused by ssDNA and dsDNA reaches as high as 70%. In order to make DNA detection fast, we also demonstrate rapid on-chip electrically enhanced hybridization using the TFTs. These technologies together will enable the realization of disposable, rapid-turnaround tools for field-deployable genomic diagnosis.     

Magnetic targeting of mechanosensors in bone cells for tissue engineering applications

Mechanical signalling plays a pivotal role in maintaining bone cell function and remodelling of the skeleton. Our previous work has highlighted the potential role of mechano-induction in tissue engineering applications. In particular, we have highlighted the potential for using magnetic particle techniques for tissue engineering applications. Previous studies have shown that manipulation of integrin attached magnetic particles leads to changes in intracellular calcium signalling within osteoblasts. However, due to the phenomenon of particle internalisation, previous studies have typically focused on short-term stimulation experiments performed within 1-2 h of particle attachment. For tissue engineering applications, bone tissue growth occurs over a period of 3-5 weeks. To date, no study has investigated the cellular responses elicited from osteoblasts over time following stimulation with internalised magnetic particles. Here, we demonstrate the long-term biocompatibility of 4.5 microm RGD-coated particles with osteoblasts up to 21 days in culture, and detail a time course of responses elicited from osteoblasts following mechanical stimulation with integrin attached magnetic particles (<2h post attachment) and internalised particles (>48h post attachment). Mechanical manipulation of both integrin attached and internalised particles were found to induce intracellular calcium signalling. It is concluded that magnetic particles offer a tool for applying controlled mechanical forces to osteoblasts, and can be used to stimulate intracellular calcium signalling over prolonged periods of time. Magnetic particle technology presents a potentially valuable tool for tissue engineering which permits the delivery of highly localised mechano-inductive forces directly to cells.     

Fabrication and characterization of a radiation sensor based on bacteriorhodopsin

Available techniques of X-ray detection have been under development due to specific shortcomings such as finite lifetime, low sensitivity, and post-processing requirements. Here we report on the fabrication of an X-ray sensor based on bacteriorhodopsin (BR) with a radius of r=3mm as the sensing area on a flexible substrate. The flexible X-ray detector can be placed on the targeted area for real-time monitoring of radiation dosage. We show that BR sensor is a potential candidate for such a powerful sensing device. For this purpose, we measure the electrical current generated by the BR sensor under different radiation dosages, energies and dose rates. This averaged current is in the range of nanoampere and is proportional to the dose rate of the received X-ray. The current also increases with the increase of radiation energy. BR radiation sensor can be readily miniaturized and is relatively easy to fabricate. The capability for real-time data collection and reusability are other advantages of this radiation sensor.     

Comparison of a potentiometric and a micromechanical triglyceride biosensor

Sensitive biosensors for detection of triglyceride concentration are important. In this paper we report on two types of silicon based triglyceride sensors: an electrolyte-insulator-semiconductor capacitor (EISCAP) which is a potentiometric device and a polysilicon microcantilever. The detection principle for both sensors is based on the enzymatic hydrolysis of triglyceride though the sensing mechanisms are different: electronic for the EISCAP and mechanical for the microcantilever. The characteristics and performances of the two sensors are critically compared. The EISCAP sensor necessitates the presence of a buffer for stable measurements which limits the sensitivity of the sensor at low concentrations of the bioanalyte to 1mM. The cantilever sensor works without a buffer which improves the lower level of sensitivity to 10 microm. Both sensors are found to give reproducible and reliable results.     

The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging

Two-photon microscopy is indispensable for deep tissue and intravital imaging. However, current technology based on single-beam point scanning has reached sensitivity and speed limits because higher performance requires higher laser power leading to sample degradation. We utilize a multifocal scanhead splitting a laser beam into a line of 64 foci, allowing sample illumination in real time at full laser power. This technology requires charge-coupled device field detection in contrast to conventional detection by photomultipliers. A comparison of the optical performance of both setups shows functional equivalence in every measurable parameter down to penetration depths of 200 microm, where most actual experiments are executed. The advantage of photomultiplier detection materializes at imaging depths >300 microm because of their better signal/noise ratio, whereas only charge-coupled devices allow real-time detection of rapid processes (here blood flow). We also find that the point-spread function of both devices strongly depends on tissue constitution and penetration depth. However, employment of a depth-corrected point-spread function allows three-dimensional deconvolution of deep-tissue data up to an image quality resembling surface detection.     

Signal amplification on planar and gel-type sensor surfaces in surface plasmon resonance-based detection of prostate-specific antigen

This article describes surface plasmon resonance (SPR)-based detection of prostate-specific antigen (PSA), comparing amplification with colloidal gold (10nm diameter) and latex microspheres (120 nm diameter) on planar- and gel-type sensor surfaces. As matrix, 3% BSA in PBS was used. Experimental data were compared with model calculations that predict the SPR signal that results from covering of the different sensor surfaces with each of the particles used. Amplification with latex particles gave a higher signal than did that with colloidal gold. However, the limit of detection (LOD) attained by latex amplification was not as good as that obtained after gold amplification, and this was unexpected. LOD and sensitivity of the amplified PSA assays when performed with the planar-type sensor disc were equally good or better compared with those when performed with the gel-type sensor disc. Indirect evidence indicates a restricted accessibility of the gel layer on the gel-type sensor toward the colloidal gold. Application of colloidal gold led to a sensitivity increase of approximately three orders of magnitude compared with nonamplified detection. The corresponding LOD was approximately 0.15 ng PSA/ml, which is sufficient for measuring enhanced, clinically relevant PSA levels (>4 ng/ml).     

Preference of food particle size among several urban ant species

Appropriate particle size may be a critical characteristic for effective granular ant baits. We examined the particle size preference of six species of pest ants to an anchovy-based bait. We also examined head capsule widths of Argentine ants, Linepithema humile (Mayr) (mean = 0.54 mm), California harvester ants, Pogonomyrmex californicus (Buckley) (mean = 1.63 mm), red imported fire ants, Solenopsis invicta Buren (mean = 0.9 mm), and southern fire ants, Solenopsis xyloni McCook (mean = 0.76 mm) and compared them with the first and second most preferred particle size. There were differences between particle size of which the most mass was removed and of which there were more particles removed by ants. California Argentine ants, southern fire ants, and Alabama Argentine ants removed more 840 to 1,000-microm particle mass of the anchovy diet but had more visits to dishes containing 420 to 590 microm particles. California harvester ants and Allegheny mound ants, Formica spp., removed more >2,000 microm particle mass but visited dishes containing 1,000 to 2,000 microm particles more often. Red imported fire ants also removed more >2,000 microm particle mass but visited dishes with 590 to 840-microm particles most often. Pharaoh ants, Monomorium pharaonis (L.), removed and visited 420 to 590-microm particles more than any other size. A linear regression model determined that particle size preferred by each ant species relates to forager head width. The majority of particles of commercial ant bait, including Amdro, Ascend, Award, Bushwhacker, Max Force with fipronil, and old and new formulations of Max Force with hydramethylnon, were 1,000 to 2,000 microm, but the majority of Niban particles were <420 microm. Altering the size of particles of toxic ant baits to fit the particle size preference of each pest ant species may increase the efficacy of ant baits.     

Feasibility of using real-time optical methods for detecting the presence of viable bacteria aerosols at low concentrations in clean room environments

An experimental investigation was carried out to determine the agreement between two methods of viable bacteria aerosol detection. Various amounts of Bacillus globigii (BG) spores were aerosolized in 1-s bursts into a HEPA-filtered air stream and sampled simultaneously with a fluorescence aerosol particle sensor (FLAPS) and a slit to agar biological air sampler. The slit sampler incorporated 150-mm malt extract culture plates, which were incubated at 37°C for at least 12 h before culturable BG particles were counted in terms of colony-forming units (CFU). A relationship between CFU and optically detected viable bacteria particles was determined as culturable particle concentrations decreased. Through further analytical procedures, the FLAPS showed a limit of detection (LOD) of 4.2 bacterial particle/2.5 l of sampled air or 1.7 × 10³ m⁻³. This real-time bacteria aerosol monitor could be used to detect burst contamination events during a surgical procedure. The technology may be used for developing a dose–response relationship between bacterial particle exposure and infection, a tool potentially helpful in determining patient risk.     

Electrical detection of human immunoglobulins G from human serum using a microbiosensor

A biosensor for the electrical detection of human antibodies from serum has been fabricated and experimentally demonstrated. The device is based on the immobilization of proteins used as probes between a set of microelectrodes. Incubation with diluted human serum was followed by incubation with anti-human secondary antibodies labeled with gold nanoparticles (GNPs) and then precipitation of silver on the nanoparticles. The output of the device was defined as the percentage of short-circuited microelectrodes after silver deposition independently of the gap conductance. Two model probes were studied: protein A and goat antibodies. The effects of the microgap spacing (5, 10, 15 or 20 microm) and the duration of the silver treatment were examined. The data obtained showed that a large spacing (20 microm) led to poor sensitivity. Alternately, 5 microm gaps led to high sensitivity and saturation of the signal. Interestingly, 10-15 microm gaps enabled a non-saturated and distinct signal for both probes that was correlated with the GNP density between the microgaps as determined by atomic force microscopy. Different capture efficiencies could be easily distinguished. The biosensor described here is easy to use and thus can be applied to real detection experiments.     

Influence of radiation quality on the yield of DNA strand breaks in SV40 DNA irradiated in solution.

Using highly energetic particles to irradiate plasmid DNA in aerobic aqueous solution, we have compiled an extensive database on how yields of DNA single- and double-strand breaks (SSBs and DSBs) vary with radiation quality. This study was performed in a low-scavenging buffer system and covers a wide range of ion species (helium to uranium) and LETs (5 to 16,000 keV/microm). For LETs up to around 40 keV/microm for SSBs and 400 keV/microm for DSBs, the total energy deposition determines cross section. At higher LET, cross sections level off and individual plateaus for particles of different atomic numbers are observed. For each ion species this is more pronounced and occurs at lower LET for SSBs than for DSBs, leading to an increase in the DSB:SSB ratio from 1:70 for X rays to 1:6 at 500 keV/microm. At this LET, the influence of track structure becomes evident, with high local concentrations of ionization events favoring the formation of DSBs and also intratrack recombination reactions. For lower-energy ions, a saturation in production of measurable DSBs is apparent, due to correlated lesion induction within densely ionizing particle tracks. For very heavy low-energy ions, both SSB and DSB cross sections decrease with particle velocity at nearly constant LET, forming individual hooked curves when plotted as a function of LET.