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Posts Tagged ‘Single-molecule detection’


From AAAS Science News on COVID19: New CRISPR based diagnostic may shorten testing time to 5 minutes

Reporter: Stephen J. Williams, Ph.D.

 

 

 

 

 

 

 

 

 

A new CRISPR-based diagnostic could shorten wait times for coronavirus tests.

 

 

New test detects coronavirus in just 5 minutes

By Robert F. ServiceOct. 8, 2020 , 3:45 PM

Science’s COVID-19 reporting is supported by the Pulitzer Center and the Heising-Simons Foundation.

 

Researchers have used CRISPR gene-editing technology to come up with a test that detects the pandemic coronavirus in just 5 minutes. The diagnostic doesn’t require expensive lab equipment to run and could potentially be deployed at doctor’s offices, schools, and office buildings.

“It looks like they have a really rock-solid test,” says Max Wilson, a molecular biologist at the University of California (UC), Santa Barbara. “It’s really quite elegant.”

CRISPR diagnostics are just one way researchers are trying to speed coronavirus testing. The new test is the fastest CRISPR-based diagnostic yet. In May, for example, two teams reported creating CRISPR-based coronavirus tests that could detect the virus in about an hour, much faster than the 24 hours needed for conventional coronavirus diagnostic tests.CRISPR tests work by identifying a sequence of RNA—about 20 RNA bases long—that is unique to SARS-CoV-2. They do so by creating a “guide” RNA that is complementary to the target RNA sequence and, thus, will bind to it in solution. When the guide binds to its target, the CRISPR tool’s Cas13 “scissors” enzyme turns on and cuts apart any nearby single-stranded RNA. These cuts release a separately introduced fluorescent particle in the test solution. When the sample is then hit with a burst of laser light, the released fluorescent particles light up, signaling the presence of the virus. These initial CRISPR tests, however, required researchers to first amplify any potential viral RNA before running it through the diagnostic to increase their odds of spotting a signal. That added complexity, cost, and time, and put a strain on scarce chemical reagents. Now, researchers led by Jennifer Doudna, who won a share of this year’s Nobel Prize in Chemistry yesterday for her co-discovery of CRISPR, report creating a novel CRISPR diagnostic that doesn’t amplify coronavirus RNA. Instead, Doudna and her colleagues spent months testing hundreds of guide RNAs to find multiple guides that work in tandem to increase the sensitivity of the test.

In a new preprint, the researchers report that with a single guide RNA, they could detect as few as 100,000 viruses per microliter of solution. And if they add a second guide RNA, they can detect as few as 100 viruses per microliter.

That’s still not as good as the conventional coronavirus diagnostic setup, which uses expensive lab-based machines to track the virus down to one virus per microliter, says Melanie Ott, a virologist at UC San Francisco who helped lead the project with Doudna. However, she says, the new setup was able to accurately identify a batch of five positive clinical samples with perfect accuracy in just 5 minutes per test, whereas the standard test can take 1 day or more to return results.

The new test has another key advantage, Wilson says: quantifying a sample’s amount of virus. When standard coronavirus tests amplify the virus’ genetic material in order to detect it, this changes the amount of genetic material present—and thus wipes out any chance of precisely quantifying just how much virus is in the sample.

By contrast, Ott’s and Doudna’s team found that the strength of the fluorescent signal was proportional to the amount of virus in their sample. That revealed not just whether a sample was positive, but also how much virus a patient had. That information can help doctors tailor treatment decisions to each patient’s condition, Wilson says.

Doudna and Ott say they and their colleagues are now working to validate their test setup and are looking into how to commercialize it.

Posted in:

doi:10.1126/science.abf1752

Robert F. Service

Bob is a news reporter for Science in Portland, Oregon, covering chemistry, materials science, and energy stories.

 

Source: https://www.sciencemag.org/news/2020/10/new-test-detects-coronavirus-just-5-minutes

Other articles on CRISPR and COVID19 can be found on our Coronavirus Portal and the following articles:

The Nobel Prize in Chemistry 2020: Emmanuelle Charpentier & Jennifer A. Doudna
The University of California has a proud legacy of winning Nobel Prizes, 68 faculty and staff have been awarded 69 Nobel Prizes.
Toaster Sized Machine Detects COVID-19
Study with important implications when considering widespread serological testing, Ab protection against re-infection with SARS-CoV-2 and the durability of vaccine protection

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Curator: Tilda Barliya PhD

Original article by: Philip Tinnefeld (2)

The ability to detect a single chemical at extremely low concentrations and high contamination is vital for earlier disease diagnosis (I). Detecting a single chemical does not only help to detect disease earlier, it is also vital for toxin/drug screening, athlete drug monitoring, environmental surveillance, and homeland security. 

To  detect a single molecule the observation volume must host only one molecule of  interest during the measurement (2). It is impossible to carry out single-molecule detection at concentrations exceeding the picomolar to low-nanomolar range, because the observation volume becomes populated by more than one fluorescent molecule as concentration increases.

This concentration barrier is crucial when using a Single-Molecule Technology such as:

  • protein-protein interactions
  • Enzyme-substrate analysis
  • DNA sequencing
  • etc

Jérôme Wenger and colleagues at AixMarseille Université in France, alongside collaborators at the ICFO-Institut de Ciencies Fotoniques and ICREA-Institució  Catalana de Recerca i Estudis Avançats in Spain, describe a system that brings two complementary nanophotonic approaches  together to achieve single-molecule detection with a high signal-to-noise ratio at a micromolar concentration (1, 2).

In recent years, two alternative nanophotonic approaches were used:

  1. Light is confined to the near-field using nanoapertures of subwavelength dimensions (typically holes in a thin aluminum or gold film on a silica substrate) that do not allow light to propagate through.  Figure 1a
  2. Enhance the excitation field  locally using a nanoantenna that converts  freely propagating optical radiation into localized energy in the form of surface plasmons.  By reducing the volume of the hot spot, it is possible to obtain enormous fluorescence enhancements up to more than 1,000-fold when compared with the case of an isolated fluorophore in solution Figure 1b

The first approach (Figure 1a)  limitations were that although this approach reduces the observation volume and enables single-molecule detection in the micromolar range, further reduction of nanoaperture size has detrimental effects on the fluorescence signal because of the decreased excitation intensity and fluorescence quenching caused by the metal film.

It did however, has some encouraging results: This method has been used, for instance, to monitor the activity of the 
enzyme polymerase1 and follow DNA sequencing in real time; and for visualizing a ribosome in action (3).

The second approach (Figure 1b) limitations were that this approach suffers from the background fluorescence of molecules far away from the hot spot but still within the diffraction-limited excited area.

Nanophotonic approach to enhance single molecule detection

nanoantenna-in-box

Wenger and colleagues synergistically combine these two approaches and place a nanoantenna within the spatial confinement of a nanoaperture (Fig. 1c), to which they called “nanoantenna-in the-box.

To demonstrate the capability of their device, they cover it with a solution containing a fluorescent dye and analyse the fluorescence signal coming out from the hot spot using fluorescence correlation spectroscopy (FCS).

The FCS technique measures the number of molecules in the effective observation volume as these diffuse in and out of it, as well as the brightness per molecule.

Limitations:

1. There are, however, a few limitations that  have to be considered. Further reduction of the gap size may result in gaps being too small for biomolecules to enter.

2. The antenna-in-box device exhibits a large surface-to-volume ratio and sticky molecules might adsorb onto the gold surface, especially in the gap, which might lead to rapid surface degradation, surfaceinduced artefacts and other background.

To overcome some of the limitations, the authors used an approach that takes advantage of a recently developed DNA-scaffolded, gap-nanoantenna might represent a viable solution. The two nanoparticles forming the gap-nanoantenna are attached to a self-assembled DNA nanostructure that provides handles to place the biomolecule of interest in the hot spot.

In summary:

“Whatever direction the research field might take, it seems as though the development of sophisticated coverslips in which nanophotonic structures such as the antenna-in-box set-up are incorporated may unlock more potential for single- molecule detection than developing more powerful microscopes”.

Ref:

I. By: Grace Rattue. Nanotechnology For Detecting Diseases Earlier.  http://www.medicalnewstoday.com/articles/245820.php

1. Deep Punj, Mathieu Mivelle, Satish Babu Moparthi, Thomas S. van Zanten, Hervé Rigneault, Niek F. van Hulst, María F. García-Parajó & Jérôme Wenger. A plasmonic ‘antenna-in-box’ platform for enhanced single-molecule analysis at micromolar concentrations. Nature Nanotechnology 8, 512–516 (2013) doi:10.1038/nnano.2013.98. http://www.nature.com/nnano/journal/v8/n7/full/nnano.2013.98.html?WT.ec_id=NNANO-201307

2. By: Philip Tinnefeld. Single-Molecule Detection: Breaking the concentration barrier.  http://www.nature.com/nnano/journal/v8/n7/full/nnano.2013.122.html

3. Sotaro Uemura.,  Colin Echeverría Aitken., , Jonas Korlach.,  Benjamin A. Flusberg., Stephen W. Turner & Joseph D. Puglisi. Real-time tRNA transit on single translating ribosomes at codon resolution. Nature 464, 1012–1017 (2010).  http://www.nature.com/nature/journal/v464/n7291/full/nature08925.html

4. G. P. Acuna, F. M. Möller, P. Holzmeister, S. Beater, B. Lalkens, P. Tinnefeld. Fluorescence Enhancement at Docking Sites of DNA-Directed Self-Assembled Nanoantennas.  Science 26 October 2012: Vol. 338 no. 6106 pp. 506-510.  http://www.sciencemag.org/content/338/6106/506.abstract

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