A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.

     

A portable surface plasmon resonance sensor system for real-time monitoring of small to large analytes

Many environmental applications exist for biosensors capable of providing real-time analyses. One pressing current need is monitoring for agents of chemical- and bio-terrorism. These applications require systems that can rapidly detect small organics including nerve agents, toxic proteins, viruses, spores and whole microbes. A second area of application is monitoring for environmental pollutants. Processing of grab samples through chemical laboratories requires significant time delays in the analyses, preventing the rapid mapping and cleanup of chemical spills. The current state of development of miniaturized, integrated surface plasmon resonance (SPR) sensor elements has allowed for the development of inexpensive, portable biosensor systems capable of the simultaneous analysis of multiple analytes. Most of the detection protocols make use of antibodies immobilized on the sensor surface. The Spreeta 2000 SPR biosensor elements manufactured by Texas Instruments provide three channels for each sensor element in the system. A temperature-controlled two-element system that monitors for six analytes is currently in use, and development of an eight element sensor system capable of monitoring up to 24 different analytes will be completed in the near future. Protein toxins can be directly detected and quantified in the low picomolar range. Elimination of false positives and increased sensitivity is provided by secondary antibodies with specificity for different target epitopes, and by sensor element redundancy. Inclusion of more than a single amplification step can push the sensitivity of toxic protein detection to femtomolar levels. The same types of direct detection and amplification protocols are used to monitor for viruses and whole bacteria or spores. Special protocols are required for the detection of small molecules. Either a competition type assay where the presence of analyte inhibits the binding of antibodies to surface-immobilized analyte, or a displacement assay, where antibodies bound to analyte on the sensor surface are displaced by free analyte, can be used. The small molecule detection assays vary in sensitivity from the low micromolar range to the high picomolar.     

Bond-rupture immunosensors--a review

It has long been the goal of researchers to develop fast and reliable point-of-care alternatives to existing lab-based tests. A viable point-of-care biosensor is fast, reliable, simple, cost-effective, and detects low concentrations of the target analyte. The target of biosensors is biological such as bacteria or virus and as such, the antibody-antigen bond derived from the real immune response is used. Biosensor applications include lab-based tests for the purposes of diagnostics, drug discovery, and research. Additional applications include environmental, food, and agricultural monitoring. The main merits of the bond-rupture method are quick, simple, and capable of discriminating between specific and non-specific interactions. The separation of specific and non-specific bonds is important for working in real-life complex serums such as blood. The bond-rupture technique can provide both qualitative results, the detection of a target, and quantitative results, the concentration of target. Bond-rupture achieves this by a label-free method requiring no pre-processing of the analyte. A piezoelectric transducer such as the quartz crystal microbalance (QCM) shakes the bound particles free from the surface. Other transducers such as Surface Acoustic Wave (SAW) are also considered. The rupture of the bonds is detected as electronic noise. This review article links diverse research areas to build a picture of a field still in development.     

Sequential detection of Salmonella typhimurium and Bacillus anthracis spores using magnetoelastic biosensors

Multiple phage-based magnetoelastic (ME) biosensors were simultaneously monitored for the detection of different biological pathogens that were sequentially introduced to the measurement system. The biosensors were formed by immobilizing phage and 1mg/ml BSA (blocking agent) onto the magnetoelastic resonator's surface. The detection system included a reference sensor as a control, an E2 phage-coated sensor specific to S. typhimurium, and a JRB7 phage-coated sensor specific to B. anthracis spores. The sensors were free standing during the test, being held in place by a magnetic field. Upon sequential exposure to single pathogenic solutions, only the biosensor coated with the corresponding specific phage responded. As the cells/spores were captured by the specific phage-coated sensor, the mass of the sensor increased, resulting in a decrease in the sensor's resonance frequency. Additionally, non-specific binding was effectively eliminated by BSA blocking and was verified by the reference sensor, which showed no frequency shift. Scanning electron microscopy was used to visually verify the interaction of each biosensor with its target analyte. The results demonstrate that multiple magnetoelastic sensors may be simultaneously monitored to detect specifically targeted pathogenic species with good selectivity. This research is the first stage of an ongoing effort to simultaneously detect the presence of multiple pathogens in a complex analyte.     

The Nucleocapsid Domain Is Responsible for the Ability of Spleen Necrosis Virus (SNV) Gag Polyprotein To Package both SNV and Murine Leukemia Virus RNA

Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimeric gag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Psi) and another viral vector RNA containing the SNV packaging signal (E). The chimeric gag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Psi. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLV pol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNV cis-acting elements are not ideal substrates for MLV pol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and other cis-acting elements.     

Temporal and Spatial Analysis of Sin Nombre Virus Quasispecies in Naturally Infected Rodents

Sin Nombre virus (SNV) is thought to establish a persistent infection in its natural reservoir, the deer mouse (Peromyscus maniculatus), despite a strong host immune response. SNV-specific neutralizing antibodies were routinely detected in deer mice which maintained virus RNA in the blood and lungs. To determine whether viral diversity played a role in SNV persistence and immune escape in deer mice, we measured the prevalence of virus quasispecies in infected rodents over time in a natural setting. Mark-recapture studies provided serial blood samples from naturally infected deer mice, which were sequentially analyzed for SNV diversity. Viral RNA was detected over a period of months in these rodents in the presence of circulating antibodies specific for SNV. Nucleotide and amino acid substitutions were observed in viral clones from all time points analyzed, including changes in the immunodominant domain of glycoprotein 1 and the 3' small segment noncoding region of the genome. Viral RNA was also detected in seven different organs of sacrificed deer mice. Analysis of organ-specific viral clones revealed major disparities in the level of viral diversity between organs, specifically between the spleen (high diversity) and the lung and liver (low diversity). These results demonstrate the ability of SNV to mutate and generate quasispecies in vivo, which may have implications for viral persistence and possible escape from the host immune system.     

An interleukin-6 ZnO/SiO(2)/Si surface acoustic wave biosensor

A novel high sensitivity ZnO/SiO(2)/Si Love mode surface acoustic wave (SAW) biosensor for the detection of interleukin-6 (IL-6), is reported. The biosensors operating at 747.7MHz and 1.586GHz were functionalized by immobilizing the monoclonal IL-6 antibody onto the ZnO biosensor surface both through direct surface adsorption and through covalent binding on gluteraldehyde. The morphology of the IL-6 antibody-protein complex was studied using scanning electron microscopy (SEM), and the mass of the IL-6 protein immobilized on the surface was measured from the frequency shift of the SAW resonator biosensor. The biosensor was shown to have extended linearity, which was observed to improve with higher sensor frequency and for IL-6 immobilization through the monoclonal antibody. Preliminary results of biosensor measurements of low levels of IL-6 in normal human serum are reported. The biosensor can be fully integrated with CMOS Si chips and developed as a portable real time detection system for the interleukin family of proteins in human serum.     

Assessing the feasibility of using neural precursor cells and peripheral blood mononuclear cells for detection of bioactive Sindbis virus

Viruses form a significant class of bio-threat agents. Currently, the only method to determine the bioactivity of viruses in vitro is to measure viral and cellular responses after co-incubation of cells with virus. Our goal is to find biomarkers for classification of agents, establishment of bioactivity, and/or prediction of disease outcomes. To begin development of a cell-based biosensor for detection of bioactive Sindbis virus (SV), our model analyte, we surveyed the outcomes of SV interaction with primary rat neural precursor cells (NPC) and human peripheral blood mononuclear cells (PBMC). Confocal fluorescence analysis of NPC treated with recombinant SV carrying green-fluorescent-protein (SV-GFP) showed that most cells were GFP positive by day 1 post inoculation. 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining of the nucleus showed nuclear condensation and fragmentation, and the percentage of TUNEL positive cells were higher in virus-treated cells than in mock-treated control. Also, there were less BrdU positive cells in virus-treated cells compared to control. Thus, SV infects NPC, decreases cellular proliferation, and induces cell death via apoptosis. PBMC were treated with SV- or UV-inactivated SV. By day 5 post infection, there were fewer adherent cells in SV-treated PBMC compared to UV-inactivated SV treated PBMC. However, the percentage of viable cells remained the same, and virus growth curves showed only clearance of virus. Thus, SV induces detachment of a subpopulation of PBMC while not killing most of the cells. Together, these results indicate that NPC and PBMC respond to bioactive SV inoculation, suggesting potential use as detectors of SV in cell-based biosensor paradigm. These studies also provide the rationale, time-scale, and phenotypic correlates for further studies with gene expression arrays.     

Characterization of immobilization methods for African swine fever virus protein and antibodies with a piezoelectric immunosensor

A direct piezoelectric flow injection analysis immunoassay for the detection of African Swine Fever virus and antibodies is presented. The peptide-specific monoclonal antibody 18BG3 and the virus protein 73 were used for detection with a quartz crystal microbalance. Accumulation of the analyte on the surface of this mass-sensitive biosensor resulted in a shift of the resonant frequency. Highly selective receptor layers were applied on the sensing electrode of the quartz crystal for detection of the complementary analyte. Different immobilization methods proved to be appropriate for coating of the monoclonal antibody 18BG3. A quartz crystal covalently coated with the antibody 18BG3 detected virus protein VP73 samples more than 20 times and was stable for more than 30 days. The coating of virus protein was performed by physisorption. A sensor with a virus protein receptor layer detected antibody 18BG3 samples 10 times within one day. The sensor device was able to perform one measurement cycle including blocking and regeneration within 30 min. With the help of a suitable carrier liquid, measurements with serum samples were performed. The calibration curves for measurements in buffer and in serum could be determined and the detection limits for virus protein detection were 0.31 and 1 μg/ml, and for antibody detection 0.1 and 0.2 μg/ml, respectively.     

Optical flow-cell multichannel immunosensor for the detection of biological warfare agents

An automated optical flow cell multichannel immunosensor for the detection and identification of toxins, viruses and bacterial particles is presented. A solid phase ELISA, based on a peroxidase label for signal generation and on fused silica capillaries as a support for immobilized antibodies, has been employed for analyte detection and identification. The sensing and signal transducing component of the sensor consists of a light-emitting diode and a photodetector. The device is fitted with three channels allowing the simultaneous detection of three agents. An integrated flow injection analysis system ensures automation of the assay cycles. Data on the detection of the bacterial toxin staphylococcal enterotoxin B (SEB), the bacteriophage M13 as a viral agent, and Escherichia coli as a bacterial agent are presented.     

Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies

Surface plasmon resonance (SPR)-based biosensors have been widely utilized for measuring interactions of a variety of molecules. Fewer examples include higher biological entities such as bacteria and viruses, and even fewer deal with plant viruses. Here, we describe the optimization of an SPR sensor chip for evaluation of the interaction of the economically relevant filamentous Potato virus Y (PVY) with monoclonal antibodies. Different virus isolates were efficiently and stably bound to a previously immobilized polyclonal antibody surface, which remained stable over subsequent injection regeneration steps. The ability of the biosensor to detect and quantify PVY particles was compared with ELISA and RT-qPCR. Stably captured virus surfaces were successfully used to explore kinetic parameters of the interaction of a panel of monoclonal antibodies with two PVY isolates representing the main viral serotypes N and O. In addition, the optimized biosensor proved to be suitable for evaluating whether two given monoclonal antibodies compete for the same epitope within the viral particle surface. The strategy proposed in this work can help to improve existing serologic diagnostic tools that target PVY and will allow investigation of the inherent serological variability of the virus and exploration for new interactions of PVY particles with other proteins.     

Nanoporous impedemetric biosensor for detection of trace atrazine from water samples

Trace contamination of ground water sources has been a problem ever since the introduction of high-soil-mobility pesticides, one such example is atrazine. In this paper we present a novel nanoporous portable bio-sensing device that can identify trace contamination of atrazine through a label-free assay. We have designed a pesticide sensor comprising of a nanoporous alumina membrane integrated with printed circuit board platform. Nanoporous alumina in the biosensor device generates a high density array of nanoscale confined spaces. By leveraging the size based immobilization of atrazine small molecules we have designed electrochemical impedance spectroscopy based biosensor to detect trace amounts of atrazine. We have calibrated the sensor using phosphate buffered saline and demonstrated trace detection from river and bottled drinking water samples. The limit of detection in all the three cases was in the femtogram/mL (fg/mL) (parts-per-trillion) regime with a dynamic range of detection spanning from 10 fg/mL to 1 ng/mL (0.01 ppt to 1 ppm). The selectivity of the device was tested using a competing pesticide; malathion and selectivity in detection was observed in the fg/mL regime in all the three cases.     

Detection of Fusarium culmorum in wheat by a surface plasmon resonance-based DNA sensor

A surface plasmon resonance (SPR) sensor based on DNA hybridization has been developed for the detection of Fusarium culmorum, a fungal pathogen of cereals. A 0.57 kbp DNA fragment of F. culmorum was amplified by specific primers and a 25-mer oligonucleotide probe was selected within the sequence of the PCR amplicon. After biotinilation, the probe was immobilized on a streptavidin sensor chip and tested for biospecific interaction with PCR products of F. culmorum. The effect of denaturating agents (formamide and urea) and ionic strength (NaCl) on hybridization efficiency of double-stranded PCR products with the immobilized probe and the specificity of the probe were investigated. The SPR biosensor was successfully used for the detection of F. culmorum in culture material of different strains and in naturally infected wheat samples. Tested on fungal cultures, it showed a good selectivity for F. culmorum against other species of either Fusarium or other fungal genera. A background signal was observed in wheat samples strictly depending on the DNA amount of the testing matrix. Testing 30 ng of durum wheat DNA the detection limit was 0.06 pg of F. culmorum DNA. The developed PCR-SPR assay allowed to detect F. culmorum with sensitivity and specificity higher than gel-electrophoresis analysis.     

A comparison of protocols for the optimisation of detection of bacteria using a surface acoustic wave (SAW) biosensor

A dual channel surface acoustic wave (SAW) device has been used as a biosensor to detect two different microorganisms, Legionella and Escherichia coli, simultaneously. A series of experiments was conducted to optimise the use of the SAW for bacterial detection using a novel protocol of coating bacteria on the sensor surface prior to addition of the antibody. Results were compared with an experiment in which a conventional protocol was utilised, where antibody was coated on the sensor surface prior to exposure to bacteria. The concentration of bacteria that attached to the surface of the SAW device was related to the antibody that specifically bound to it and therefore to frequency in a dose dependent fashion. Unlike conventional microbiological techniques quantitative results can be obtained for Legionella and E. coli down to 10(6) cells per ml within 3 h. In addition E. coli was detected down to 10(5) cells per ml in a modified protocol using sheep IgG as a blocking agent.     

Long-term dynamics of Sin Nombre viral RNA and antibody in deer mice in Montana

Infections with hantaviruses in the natural host rodent may result in persistent, asymptomatic infections involving shedding of virus into the environment. Laboratory studies have partially characterized the acute and persistent infection by Sin Nombre virus (SNV) in its natural host, the deer mouse (Peromyscus maniculatus). However, these studies have posed questions that may best be addressed using longitudinal studies involving sequential sampling of individual wild-caught, naturally infected mice. Using enzyme immunoassay and polymerase chain reaction (PCR) analysis of monthly blood samples, we followed the infection status of deer mice in a mark-recapture study in Montana for 2 yr. Only six of 907 samples without IgG antibody to SNV contained detectable SNV RNA, suggesting that there is a very brief period of viremia before the host develops detectable antibody. The simultaneous presence of both antibody and viral RNA in blood was detected in consecutive monthly samples for as long as 3 mo. However, chronic infection was typified by alternating characteristics of PCR positivity and PCR negativity. Two possible interpretations of these results are that 1) viral RNA may be consistently present in the blood of chronically infected deer mouse, but that viral RNA is near the limits of PCR detectability or 2) SNV RNA sporadically appears in blood as a consequence of unknown physiological events. The occurrence of seasonal patterns in the proportion of samples that contains antibody and that also contained SNV RNA demonstrated a temporal association between recent infection (antibody acquisition) and presence of viral RNA in blood.     

P450-based porous silicon biosensor for arachidonic acid detection

A porous silicon biosensor based on P450 enzyme for arachidonic acid detection was developed. A new transduction method is presented with a simultaneous measurement of refractive index and fluorescence intensity changes when the analyte is binding to an enzyme on the porous silicon surface. A fluorophore bound to a cysteine residue in an allosteric position of the haem domain (BMP) of cytochrome P450 BM3 enhances its fluorescence intensity upon interaction with its substrate arachidonic acid, involved in diseases such as Alzheimer's, liver cancer and cellular inflammation processes. BMP has been anchored on porous silicon surface and the new transduction method has been successfully exploited to develop a biosensor for arachidonic acid, reaching a detection limit of 10 μM arachidonic acid in a dynamic range of 10-200 μM. Moreover, the change of the refractive index has been also monitored at the same time, displaying a higher detection limit of 30 μM. Preliminary test were also conducted in plasma proving the high specificity and selectivity of the sensor even in presence of interferents in the range of 50-100 μM. Here we suggest these two detection systems could be used simultaneously to increase the accuracy and the dynamic range of the sensor avoiding a false positive response.     

The BARC biosensor applied to the detection of biological warfare agents

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.     

Gas phase activity of anti-FITC antibodies immobilized on a surface acoustic wave resonator device

In this paper we present the results of a series of experiments on the activity of antibodies in a vapor phase sensor. For these experiments the sensor component was a ST-Quartz resonator with a center frequency of approximately 250 MHz. Anti-FITC antibodies were attached to the electrodes on the device surface via a protein-A crosslinker. Surface acoustic wave (SAW) resonator devices with various coatings were mounted in TO-8 packages, inserted into our sensor head module and subjected to various fluorescent analyte gases. Numerous controls were performed including the use of coated and uncoated devices along with devices coated with antibodies which were not specific for the target analyte. The SAW immunosensor response was monitored and a baseline frequency shift was observed when the analyte being presented was the antigen for the immobilized antibody. To provide an independent measure of antibody/antigen binding, the devices were removed from the sensor head, washed with a buffer solution to remove any unbound analyte, and then inspected using a confocal laser scanning microscope (CLSM). Since all the analytes being used in these experiments were fluorescent this afforded us the opportunity to visualize the attachment of the analyte to the antibody film. Given the high resolution of the CLSM, we were able to identify the location of the attachment of the fluorescent analytes relative to the 1.5 microm wide electrodes of the SAW device. We believe that these experiments demonstrate that we have achieved real time molecular recognition of these small molecules in the vapor phase.