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RESEARCH PROFILE Universal biosensor Whether it’s monkeypox, SARS, E. coli, Cryptosporidium parvum, Bacillus anthracis, you name it, each time a new organism makes the headlines, a new sensor is needed to detect it. What if you could easily make one sensor detect any pathogen of interest? That is just what Antje Baeumner and colleagues at Cornell University are hoping to do with their new universal biosensor, which can detect any known nucleic acid sequence. The generic sensor, which is described in the February 15 issue of Analytical Chemistry (pp 888–894), readily converts to a specific biosensor within minutes and offers nanomolar detection limits. “The biosensor components themselves are so generic that you can implement any nucleic acid sequence that you like in a very short incubation step,” says Baeumner. “So now somebody who is not familiar with developing biosensors could actually use our sensor, even if they are not looking at the same organisms or the nucleic acid sequences that we are,” she adds. The sensor contains two universal components—liposomal nanovesicles with generic oligonucleotide sequences and polyethersulfone membrane strips with streptavidin. To make the sensor specific to a particular DNA or RNA sequence, the liposomal nanovesicles are incubated with a reporter probe, a capture probe, and a target sequence, thereby forming a hybridization sandwich. “You have to know the sequence you would like to detect and the sequence for the capture and reporter probes,” says Baeumner. “In addition, you have to have biotin on the capture probe and an additional sequence on the reporter probe that allows the rapid attachment to our biosensor.” One end of the reporter probe binds to the generic oligonucleotide sequence, and the other end binds to the target sequence. In addition, one end of the capture probe binds to a different segment of the target sequence. Following an in84 A
Reporter probe Target sequence
Immobilized streptavidin
Liposome with generic oligonucleotide sequences
Complex after hybridization occurs Biotinylated capture probe
In a universal biosensor assay, liposomal nanovesicles with generic oligonucleotide sequences are incubated for 20 min at 41 °C with a reporter probe, a capture probe, and a target DNA or RNA sequence. The hybridization mixture is pipetted onto a polyethersulfone membrane strip containing streptavidin and allowed to migrate to the detection zone by capillary action. The target sequence is captured in the detection zone by streptavidin–biotin binding.
cubation period, the hybridization mixture is transferred onto the membrane strip and allowed to migrate by capillary action. The biotinylated end of the capture probe binds to streptavidin in the detection zone, capturing the target sequence. The liposomal nanovesicles entrap dye molecules in such a way that the signal can be evaluated visually for qualitative purposes or with a portable reflectometer for quantitative measurements down to the nanomolar level. To test their new universal sensor, Baeumner and colleagues isolated mRNA sequences from B. anthracis, C. parvum, and E. coli. After amplifying the sequences by nucleic acid sequence-based amplification (NASBA), they analyzed them with both the universal sensor and sensors specific to each organism. In all cases, the universal biosensor matched or exceeded the performance of the specific RNA biosensors, which were previously developed in Baeumner’s laboratory. The universal sensor could be used instead of lengthy nucleic acid assays such as the Northern or Southern blot and expensive instruments such as the LightCycler, which amplifies and quantifies a given sequence all in one machine.
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“Any researcher trying to assess how much of a specific sequence they have following an amplification reaction, such as PCR or NASBA, would be able to use our biosensor,” says Baeumner. In addition, she sees microbiologists using the universal sensor to confirm whether they have a particular kind of organism without any amplification reaction. “If somebody would like to very rapidly develop a sensor against a certain pathogen, all they have to do is find the sequences,” she adds. For something like a contaminated food source, if it is highly contaminated and enough of the food is available, the sensor could detect the offending organism directly. But if there are only a few organisms per gram of food, an amplification reaction would have to be done first, she says. The researchers are currently testing the sensor on four new sequences. One is a different sequence from B. anthracis, and another is from Yersinia pestis, the microorganism that causes bubonic and pneumonic plague. Baeumner would not disclose the other two, but she did hint that one of them is a virus. a —Britt E. Erickson
