Biosensors

Abstract

BioTechniquesVol. 41, No. 4 Tech NewsOpen AccessBiosensorsLynne LedermanLynne LedermanSearch for more papers by this authorPublished Online:21 May 2018https://doi.org/10.2144/000112276AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Spying on the InvisibleBiosensors use biomolecular probes to detect the presence of biologic and chemical molecules or structures, can be based on antibody-antigen reactions, nucleic acid hybridization, protein conformation changes, or other reactions, and rely on conversion of those reactions into chemiluminescent, electrochemical, optical, or other signals for detection.Biosensors have a wide variety of currently exploited and potential applications, including (i) monitoring food products for pathogens, pesticides, and other contaminants; (ii) direct and rapid detection of infectious agents in the bloodstream or by analyzing patients’ breath; and (iii) screening people, freight, vehicles, and buildings for illegal drugs, bioterror agents, and explosives. Key features for successful biosensing systems are accuracy and an ability to detect a variety of analytes at low concentrations. Depending on the application, biosensors may be required to handle very large volumes (e.g., environmental air or water supplies) or minuscule samples (e.g., a drop of blood). Other desirable features are rapid turnaround, high-throughput, miniaturization, portability, automation, and remote operation.MicroanalysisAntje Baeumner, Associate Professor, Biological and Environmental Engineering, Cornell University, Ithaca, NY is developing microfluidic biosensors to detect mRNA and rRNA of pathogens. In applications involving food and for bioterror screening, RNA rather than DNA is the target analyte, because the goal is to detect live, rather than dead, organisms. Her work involves the development of electro-flow assays. “The platform technology is well developed,” she says, “and is now easy to adapt to new organisms.” Multianalysis is important in many applications, such as for detecting Dengue virus, in which there are four serotypes. Baeumner says her group has developed electroflow assays for all four Dengue serotypes. “It's important to distinguish infection with one serotype from infection with more than one serotype, because mortality is greatly increased if a patient has a dual infection.” They are also developing a handheld electroflow device that uses two AA batteries as a power source. In addition to virus detection, they are developing systems for Gram positive and negative bacteria and protozoans, such as Cryptosporidium parvum, which can contaminate drinking water.Prototype of a multichannel electrochemical microbiosensor, shown here filled with sulforhodamine B for the visualization of the channel structure.Image courtesy of Antje Baeumner, Cornell University, Ithaca, NY.Sample preparation requires further development. Currently, sample preparatory steps must be done at the laboratory bench. The ability to prepare samples in the field rather than sending a specimen to the laboratory is an obvious goal. “Bioterror is the reason we have so many applications. It doesn't matter where it comes from or what it is. Sample preparation is crucial,” Baeumner says. They are collaborating with a small biotech company that is looking for a third company to partner to commercialize the technology. “We expect commercial production within the next year.” A microfluidics-based total analysis system, she believes, is about 3 to 5 years away. “What makes our technology interesting is integrating high sensitivity and specificity in a simple system. Current technologies can detect pathogens rapidly if there are large numbers of organisms, but if only a few, it takes time,” she observes. Their ultimate goal is to develop a sensor when one doesn't know beforehand what needs to be detected and that is capable of detecting those unknowns at low concentration.She believes that the ceiling for electroflow detection is four to five up to eight to ten analytes at a time. They are using five for proof of principle for a microfluidic, multiparallel channel system, with an expansion target of 50 simultaneous analytes. One might not get all the unknowns one wants to detect all at one time, but it would work, certainly for food-borne pathogens, for example, those in hamburger, where there are a limited number of things to look for, like Salmonella and Escherichia coli.In clinical applications, one may have one device for viruses and another one for bacteria, because in many instances, one would be likely to know the type of agent causing the infection. “I can envision in 10 years having something like a glucose monitor that patients are now using” for infectious agent identification, she concludes.NanosensingShana O. Kelley, Professor of Chemistry, Boston College, Chestnut Hill, MA, says her group's particular interest is using nanostructures for electrochemical biosensors. “Electrochemical biosensors are cheaper than those based on optical detection. Nanostructures provide better sensitivity and detect at low levels.” Kelley says. “Nucleic acid-based biosensing makes sense as a new technology for high-throughput genetic screening. Microarrays take time and are more expensive.”Advances in the last couple of years have allowed bypassing bottlenecks to clinical diagnosis. “There's an incredibly bright future,” Kelley says. “The dream everybody has is for a chip to analyze a predisposition for cancer, instead of analyzing tumors after they occur, and catch it at an early stage.”Image 2. Electrochemical biosensors. (A) Scanning electron micrograph of gold nanowires, grown within a plastic template, that are used for electrochemical biosensing. Each nanowire is approximately 30 nm in diameter, only a factor of 10 larger than the biomolecular target it will attempt to “catch” out of a sample. (B) To be used for biosensing, probe DNA strands are attached to gold nanowires (left), then complementary target nucleic acids can be hybridized from solution (right). The presence of the target strand is detected using an electrochemical reporter system that allows very sensitive current-based measurements.Top image courtesy of Shana O. Kelley, Boston College, Chestnut Hill, MA.Like Baeumner, Kelley sees RNA as a target, but because there are potentially more copies of a given sequence, so it is easier to find. She also agrees that sample preparation can be the hardest part and, being slow, is another bottleneck. Relatively abundant or not, even RNA is present in relatively small amounts and requires special handling. In addition, biologic samples are heterogeneous, containing interfering substances. “You need to work it up to get a clean sample,” Kelley says, “but there are ways around it. Microfluidics are a great solution to the problem. Integration of microfluidic sample preparation with nanostructure electrochemical biosensors into automated systems is tough, but will happen. We have all the pieces.”As a chemist, Kelley is involved in basic research, mostly assay and technology development, and her group collaborates with chemical engineers to take their research to make functional devices. “The actual device development is an industrial problem,” she observes. Like Baeumner, Kelley also believes that within the coming decade there will be many more methods at our disposal for disease detection.On the SurfaceThe laboratory of Robert M. Corn, Professor, Departments of Chemistry and Biomedical Engineering at the University of California, Irvine, CA, uses surface plasmon resonance (SPR) imaging to detect bioaffinity interactions without relying on fluorescent labels. “None of the molecules in body are fluorescently labeled,” Corn observes. SPR imaging relies on surface bioaffinity interactions. One molecule, for example DNA, is attached to a surface or is in a film near its surface. If a second molecule, for example a protein like a transcription factor, binds to the DNA on the surface, the protein may be altered in some way, such as by changing conformation, which can then be detected. Other types of molecules that can be attached to or in the film surface include antibodies that can be used to detect biomarkers or other antigens in solution. Thin (45 nm) gold films are chemically modified, especially by surface enzyme chemistry, for SPR. The chemistry can be controlled better with gold, but silver or copper can also be used.Image 3. Surface plasmon resonance (SPR) imaging. (A) SPR imaging apparatus. SPR imaging measures the reflectivity image from a gold thin film-prism assembly. Changes in the local index of refraction due to the surface bioaffinity processes results in a differential reflectivity image that can be used to quantitatively determine biomolecules (10 kDa or larger) adsorbed from solution. (B) SPR imaging differential reflectivity images. The SPR images of microarrays can be of squares created by photopatterning and spotting or lines created with microfluidics. Feature sizes can be as small as 50 m. (C) Microfluidic arrays. Microfluidics can be used to create surface patterns of biomolecules and also to reduce the target volumes down to 1 L or less. (D) Surface enzyme chemistry. Enzyme reactions such as the RNase H hydrolysis of surface-bound heteroduplexes can be used both to enhance the sensitivity and selectivity of the SPR imaging measurements and to create novel array fabrication methodologies.Bottom image courtesy of Robert M. Corn, Professor, University of California, Irvine, CA.SPR detection is optical, using charge-coupled device (CCD) cameras, like those in cell phones. Electrical biosensors are hard to make multiplex, Corn observes, but electrochemical biosensing is cheaper than optical methods. His group is working on the next generation of biosensors, incorporating ways to making SPR imaging more sensitive. One way is to use nanoparticles to amplify the signal; another is by using surface enzyme reactions to amplify the signal. They are also incorporating a microfluidics format to allow analysis of a small sample (e.g., one drop of blood). “I'm interested in biochips and silicon technology to create arrays. I am a tools developer, a chemist, not a biologist,” he says. “Although we can detect SNPs, RNA aptamer arrays, and miRNA, we have no particular biologic goal.”Corn looks at chip technology in terms of generations. The first generation was chemical bioaffinity, where there was one molecule on the surface and one molecule in solution, and their binding was detected. This is the basis of all chip technology, he says. “We are now in the second generation, which is coming online. This is using surface enzyme chemistry, where binding events lead to reactions, giving more information.” The third generation, he believes, will allow multiple reactions at multiple sites on a chip, and will depend, in part, on the use of microfluidic technology. Corn envisions being able to put DNA in one end of a chip and get the encoded protein out at other end after multiple binding and reaction steps in a fully automated system. “We should be able to transfer all of biotechnology from Eppendorf tubes to microchips,” he predicts.FiguresReferencesRelatedDetails Vol. 41, No. 4 Follow us on social media for the latest updates Metrics Downloaded 261 times History Published online 21 May 2018 Published in print October 2006 Information© 2006 Author(s)PDF download