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.     

Detection of the toxin domoic acid from clam extracts using a portable surface plasmon resonance biosensor

The amnesic shellfish poison domoic acid is produced by marine algae of the genus Pseudo-nitzschia. We have developed a portable surface plasmon resonance (SPR) biosensor system for the detection of domoic acid. Because of concerns with domoic acid contamination of shellfish, there is a need for rapid field quantification of toxin levels in both shellfish and seawater. Antibodies were raised against domoic acid and affinity purified. These antibodies were used to develop competition- and displacement-based assays using a portable six-channel SPR system developed in our laboratories. Standard curves for detection of domoic acid in phosphate buffered saline and in diluted clam extracts analyzed by the competition-based SPR assay demonstrated a limit of detection of 3 ppb (10 nM) and a quantifiable range from 4 to 60 ppb (13–200 nM). Comparison of analyses for domoic acid levels in Pacific razor clams, Siliqua patula, containing moderate to high levels of domoic acid by the standard HPLC analysis protocol and the SPR-based assay gave an excellent correlation. This same technology should also function for detection of domoic acid in concentrated algal extracts or high dissolved levels in seawater.     

Compact, high performance surface plasmon resonance imaging system

We report the construction and characterization of a new compact surface plasmon resonance imaging instrument. Surface plasmon resonance imaging is a versatile technique for detection, quantification and visualization of biomolecular binding events which have spatial structure. The imager uses a folded light path, wide-field optics and a tilted detector to implement a high performance optical system in a volume 7 in. x 4 in. x 2 in. A bright diode light source and an image detector with fast frame rate and integrated digital signal processor enable real-time averaging of multiple images for improved signal-to-noise ratio. Operating angle of the imager is adjusted by linear translation of the light source. Imager performance is illustrated using resolution test targets, refractive index test solutions, and competition assays for the antiepileptic drug phenytoin. Microfluidic flowcells are used to enable simultaneous assay of three sample streams. Noise level of refractive index measurements was found to decrease proportional to the square root of the number of pixels averaged, reaching approximately 5 x 10(-7) refractive index units root-mean-square for 160 x 120 pixels image regions imaged for 1s. The simple, compact construction and high performance of the imager will allow the device to be readily applied to a wide range of applications.     

A Highlights from MBoC Selection: Nonmuscle myosin-2 contractility-dependent actin turnover limits the length of epithelial microvilli

Brush border microvilli enable functions that are critical for epithelial homeostasis, including solute uptake and host defense. However, the mechanisms that regulate the assembly and morphology of these protrusions are poorly understood. The parallel actin bundles that support microvilli have their pointed-end rootlets anchored in a filamentous meshwork referred to as the “terminal web.” Although classic electron microscopy studies revealed complex ultrastructure, the composition and function of the terminal web remain unclear. Here we identify nonmuscle myosin-2C (NM2C) as a component of the terminal web. NM2C is found in a dense, isotropic layer of puncta across the subapical domain, which transects the rootlets of microvillar actin bundles. Puncta are separated by ∼210 nm, the expected size of filaments formed by NM2C. In intestinal organoid cultures, the terminal web NM2C network is highly dynamic and exhibits continuous remodeling. Using pharmacological and genetic perturbations in cultured intestinal epithelial cells, we found that NM2C controls the length of growing microvilli by regulating actin turnover in a manner that requires a fully active motor domain. Our findings answer a decades-old question on the function of terminal web myosin and hold broad implications for understanding apical morphogenesis in diverse epithelial systems.     

Portable 24-analyte surface plasmon resonance instruments for rapid, versatile biodetection

Field use of surface plasmon resonance (SPR) biosensors for environmental and defense applications such as detection and identification of biological warfare agents has been hampered by lack of rugged, portable, high-performance instrumentation. To meet this need, we have developed compact multi-analyte SPR instruments based on Texas Instruments' Spreeta sensing chips. The instruments weigh 3 kg and are built into clamshell enclosures measuring 28 cm x 22 cm x 13 cm. Functions are divided between an electronics unit in the base of the box and a fluidics assembly in the lid. Automated valves and pumps implement an injection loop flow system that allows sensors to be exposed to sample, rinsed, and treated with additional reagents (such as secondary antibodies) under computer control. Injected samples flow over the surfaces of eight sensor chips fastened into a temperature-controlled silicone flowcell. Each chip has 3 sensing regions, for a total detection of 24 areas that can be simultaneously monitored by SPR. Coating these areas with appropriate antibodies or other receptors allows a sample to be screened for up to 24 different substances simultaneously. The instruments report refractive index (RI) values every second, with a typical noise level of 1-3 x 10(-6) RI units. The design of the device is described, and performance is illustrated with detection of six distinct analytes ranging from small molecules to whole microbes during the course of a single experiment.