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BioHawk Frequently Asked Questions
Is it possible to quantify the BioHawk assay analysis?
Yes. What you do is challenge the coupon with a range of concentrations of the target analyte, and in that way obtain a response curve. While the unit will not compute concentrations directly, the data can be downloaded and the data compared with the response curve. If you had a customer that was going to buy a large number of units, we could provide custom software (hopefully, paid for by the user) that allowed you to load a response curve into the BioHawk (or RAPTOR) and which would provide a numeric output. Users generally prefer to stay away from numeric outputs, as field operators usually are more interested in and capable of understanding presence/absence, rather than actual concentration.
Please explain in detail why the disposable waveguide probes can be re-used up to 15 times? Once a virus is detected does the waveguide probe need to be thrown away?
The waveguides are coated with a target-specific antibody. If the target is not present in a sample, the coating is still good, and is available for further sample challenges. The coating is stable for up to 24-48 hours, depending on temperature, the presence of chemicals that might cause the antibodies to become damaged, or bacteria that might like to eat the antibody coating. The secondary fluorescent reagent is stored and reused, so its life is long as well. It does not attach to the waveguide unless the target substance is present in the sample, so it is not depleted except through dilution. cause by mixing with water fillets in the coupon's waveguide portion.
Once one of the waveguides has captured some of its targeted substance, that particular waveguide is compromised, but the other waveguides (assuming they target something else) would still be unaffected. Subsequent assays may show a small positive response on that channel, even if there is no targeted substance present in later samples, due to the fact that the secondary antibody reaction is not 100% effective at labeling each captured target on the first use. This is a conscious choice on our part. By not going to equilibrium on the step where the waveguide is soaked in the secondary antibody, the assay time is significantly shortened.
We use sandwich assays to detect viruses, do we use the same principle to detect chemicals like Sarin?
Sarin is too small a molecule to be detected. Usually, a molecule has to have a molecular weight of over 500 to have enough surface structure to be recognized by an antibody. Sarin only has a molecular weight of 140. It might be possible to engineer non-antibody based assay reactions for analytes such as Sarin, but such an option is not currently offered.
Please explain the difference between array, coupon and recipe.
An "array" is usually a grouping of discrete chemical reaction sites.
A "coupon" is a disposable credit card-sized assay module that has fluidics and chemistry integrated together. It would have an array of capture antibody sites and fluidic channels connecting the sites to the instrument. In the BioHawk coupon, it additionally stores the secondary antibody reagent in small onboard reservoirs.
A "recipe" is a series of instrument operations that together, allow a user to perform an assay under total computer control. In general, these operations include turning pumps and valves on and off for programmed times, turning interrogating lasers on and off for programmed times, storing samples for confirmatory analysis, and performing aerosol sample collection for a programmed time.
What are the detection parameters for BioHawk?
The BioHawk measures the total fluorescent light produced by dye molecules captured onto the waveguide surface. These dye molecules are captured during the bioassay process, if the target molecule or organism was first captured by the sensing surface. There are arrays of photodiodes under the bioassay coupon. This fluorescent light, after passing through an optical filter that removes most of the exciting laser light, impinges on the photodiode arrays and is converted to photocurrents by the photodiodes. So the ultimate measured electronic parameter is an electrical current, while the physical parameter being monitored is the number of dye molecules attached to the capture surface.
Calibration is done by challenging the device with different concentrations of the target, and then getting a plot of signal versus concentration. Most users aren’t interested in quantitative results, so we parse the response into “negative,” “suspect,” “positive,” and “high positive.”
What is the life cycle of one BioHawk coupon when detection result is “positive,” “negative” or a combination of both? How do you calculate coupon life cycle? And how do you define the start and an end of a reaction?
A ‘"Channel" area set aside for each specific target. The optics are set up to focus eight different sensing areas onto eight different sets of photodiodes. Hence, the BioHawk can be set up to simultaneously monitor up to 8 "channels" or equivalently, eight different targets. If you do 10 measurements on a coupon, then the number of measurements per target is 10, but the total number of measurements for all channels is 8 x 10 = 80 separate bioassays.
The surface reactions we harness to detect targets are not run to completion because that would take too much time. Instead, we allow (through the microprocessor-controlled fluidics) the liquid sample and the reagents to contact the sensing surfaces for a precise amount of time that is less than the amount needed for the reporter reactions to go to completion. This allows an assay to be completed in 15 minutes- as compared to 0.5 hr to 1 hour if we were allowing everything to equilibrate at each step.
The number of assays that can be performed is limited by dilution of the reagent liquid onboard the coupon. We have four small reagent reservoirs of about 0.35 ml each on the coupon. Every time reagent is used inside the coupon, it is pumped out onto the sensing surfaces. After a few minutes, it is pumped back into the reservoirs. Every time you do this, small fillets of water in the sensing area mix with the reagent and dilute it a little. After 10 to 15 assays, the reagent concentration has begun to drop a little and the assay may not be as sensitive. The dilution rate is somewhat variable, so that is why you might find both 10 and 15 assays noted in different places. To be safe, you should not quote more than 10 runs per coupon.
Our target reagents include biological agents, toxins, explosives and chemical contaminants. Can one coupon hold a mixture of 4 different classes of target reagents at once?
Yes, there is no problem with different classes. But note that the classes we have assays for right now are toxins, viruses, bacteria, spores, and microorganisms; not explosives or chemicals. There are other methods for those targets that are probably better, unless there are unusual circumstances.
Is BioHawk able to detect and identify the newest flu, the H1N1?
While the air sampler portion can collect the H1N1 virus, we do not have an assay for it.
How large an area can BioHawk cover for sampling? For example, if a room has dimensions of 10 feet by 10 feet by 10 feet that needed to be cleared and how many BioHawks would need to be present to finish the clearance in one cycle (i.e 15 min.)?
If you want to sample all the air in a room once, and do it in a finite time, then you need a high volume concentration device like the SASS 4000 or SASS 4100, which collect at a rate of about 3,600 LPM. At the SASS 2300's 325 LPM, it would take over 80 minutes to do one air turn, while the SASS 4000 would do one turn in about 8 minutes. However, micron-sized pathogens will tend to diffuse and float about, even in a closed space, and it is not usually necessary to sample all the air to find an aerosol pathogen. The minimum-size air sample is instead dictated by the sensitivity of the assay used, the air concentration of the pathogen, the liquid sample size, the sampling time, and the volumetric sampling rate. Some assay methods are sensitive to the total number of pathogens captured, but most have a detection limit that can be characterized as a minimum pathogen concentration in the liquid sample. So you want to do whatever you can to increase the pathogen's concentration in the liquid. You can do this by increasing the air flow rate, so long as it does not adversely affect capture efficiency; decrease the liquid sample volume; and/or increase the sampling time.
Can we expect an extended lifetime of a coupon over 24 hours after hydration when no bio-assay is done during the period?
You may expect some prolonged usage time. However, Research International cannot assure performance after 24 hours. Usage time is prolonged if system operation is below 20° C, and is shortened at higher temperature. We have tested coupons up to 50° C, and the 24-hour lifetime is conservative. Let us know if a more detailed relationship between operating temperature and lifetime is needed.
If there are flurophore floating in the room while the assay process is taking place, will it affect the result? If not, how does the coupon keep flurophore away?
There just aren't significant amounts of fluorescent material floating around in the air that can be stimulated by 632 nm excitation light, so unless you're in a fluorescent paint factory, I don't think this issue will ever arise in real world aerosol sampling! The fluorophores used in the coupon are specially designed to efficiently fluoresce with 632 nm excitation, and there just aren't very many natural aerosols that will confound the instrument.
There may be some fluorescent algae that could be detected by the instrument through their solution fluorescence, but the instrument doesn't actually take any readings while the sample is incubating on the waveguide. The before-and-after fluorescent readings that determine whether something has been captured are only taken while the coupon is filled with clean buffer from the storage reservoir. Buffer fluid is used to flush out any fluorescent species that were present during the sample incubation step. In other words, there are no readings taken during the time the sample is resident in the coupon, so any residual fluorescent material should be at a very low level.
