Simple, inexpensive, fast and accurate nano-sensors pinpoint infectious diseases

Date:
February 2, 2022
Source:
Arizona State University
Summary:
Researchers describe a novel method for detecting viruses like
Ebola virus (EBOV) and SARS CoV-2.



FULL STORY ==========================================================================
In recent years, deadly infectious diseases, including Ebola and COVID-19,
have emerged to cause widespread human devastation. Although researchers
have developed a range of sophisticated methods to detect such infections, existing diagnostics face many limitations.


==========================================================================
In a new study, Chao Wang, a researcher at Arizona State University's
Biodesign Institute and School of Electrical, Computer & Energy
Engineering, along with ASU colleagues and collaborators at the University
of Washington (UW), Seattle describe a novel method for detecting viruses
like Ebola virus (EBOV) and SARS CoV-2.

The technique, known as Nano2RED, is a clever twist on conventional high- accuracy tests relying on complex testing protocols and expensive readout systems. The in-solution nano-sensors ("Nano2" in the name) serve to
detect disease antigens in a sample by simple mixing. The innovative
Rapid and Electronic Readout process ("RED") developed in the Wang lab
delivers test results, which are detectable as a color change in the
sample solution, and record the data through inexpensive semiconductor
elements such as LEDs and photodetectors.

The technology represents a significant advance in the fight against
infectious diseases. It can be developed and produced at very low cost, deployed within weeks or days after an outbreak, and made available for
around 1 cent per test.

Compared with widely used high-accuracy lab tests, such as ELISA, Nano2RED
is much easier to use. It does not require surface incubation or washing,
dye labelling, or amplification, yet still provides about 10 times
better sensitivity than ELISA. In addition, the use of semiconductor
devices supports a highly portable digital readout system, which can
be developed and produced at a cost as low as a few dollars, making
it ideal not only for lab use but for clinics, home use, and remote or resource-strained locations. This approach is based on modular designs,
and could potentially be used to test for any pathogen.

"This technology works not because it is complex but because it
is simple," says professor Wang. "Another unique feature is the multidisciplinary nature of biosensing. A fundamental understanding
of biochemistry, fluidics, and optoelectronics helped us come up with
something this 'simple'." Wang is a researcher with the Biodesign Center
for Molecular Design and Biomimetics at ASU. He is also a researcher
with ASU's School of Electrical, Computer and Energy Engineering; and
the Center for Photonic Innovation. Dr.

Liangcai Gu is the collaborator at Department of Biochemistry and
Institute for Protein Design at UW, Seattle.



==========================================================================
The research appears in the current issue of the journal Biosensors
and Bioelectronics. Dr. Xiahui Chen and Md Ashif Ikbal from ASU and
Dr. Shoukai Kang from UW are the first authors, and Jiawei Zuo and Yuxin
Pan are the other contributing authors.

The testing bottleneck Epidemiologists have long known the basic formula
when confronting a disease outbreak. To identify cases and stop the
contagion, it is necessary to develop an accurate test or assay that can identify the disease, then test early and often, to assess the rate of
spread and attempt to isolate the infected.

Unfortunately, by the time a new diagnostic has been developed,
manufactured, and distributed, the disease outbreak is often already
widespread and challenging to contain. Further, accurate tests including
PCR, (which can amplify tiny levels of pathogenic nucleic acids to
measurable levels), are often expensive, labor-intensive and require sophisticated laboratory facilities.

The Ebola epidemic of 2014-2016, though largely confined to West Africa,
spread with terrifying speed, causing panic and killing more than 11,000 people. The virus' rate of lethality, one of the highest for any known pathogen, can exceed 90%, depending on disease strain. The crisis was exacerbated by a combination of inadequate surveillance systems and poor
public health infrastructure.



==========================================================================
SARS CoV-2, though less lethal than Ebola, has spread to every
country on earth and has already killed more than 5.6 million people
worldwide. In both disease outbreaks, diagnostic testing arrived late
on the scene. Further, costly and cumbersome testing requirements have
meant that far too few tests have been administered, even after their successful development.

The new study applies its innovative method to test for these two
prominent diseases, as a proof of concept.

Tidal wave A common feature in many disease outbreaks is the lightning
speed with which a pathogen, having first infected a handful of people,
can gather momentum, fan out in all directions and quickly overwhelm
hospitals and healthcare providers.

Cutting off a pathogen's routes of transmission requires identifying
and isolating sick individuals through testing, as quickly as possible.

During a pandemic like COVID-19, the sensitivity of a given diagnostic
test is secondary to how often the test is given and how long it takes
for results to be processed. A highly sensitive test is of limited
use if it can only be given once, and results require a weeks-long
turnaround. Research has shown that infection outbreaks are best
controlled when testing is repeated in less than 3-day intervals and at
a large scale.

Adequately preparing society for current and future outbreaks of
infectious disease will require faster, cheaper, more accurate and more
easily usable diagnostics.

Close affinity The new technology can identify secreted glycoprotein
(sGP), a telltale fingerprint of Ebola virus disease and the SARS-CoV-2
spike protein receptor binding domain (RBD). The technology is highly
accurate, rivaling ELISA, a long-recognized gold standard technology
for diagnostic testing.

The basic idea of such diagnostics, known as immunoassays, is simple:
a sample of blood (or other biological fluid) is applied to the assay,
which is adorned with antibodies. When antibodies recognize the presence
of a corresponding disease antigen in the sample, they bind with it,
producing a positive test result. In the ELISA test, the disease antigen
needs to be immobilized on a flat surface.

Nano2RED also relies on binding affinity for positive diagnosis
but instead uses floating gold nanoparticles for readout. Unlike
ELISA, Nano2RED can be developed from scratch in roughly 10 days and theoretically applicable for any pathogen, providing vitally important
early surveillance in the case of a disease outbreak. It can deliver
test results in 15-20 minutes and may be administered at an estimated
cost of a penny per test. In the current study, the new test was shown
to detect Ebola's sGP in serum with a sensitivity roughly 10 times better
than ELISA.

Sensing danger The accompanying graphic shows how the method works:
The first step is to produce a very large library containing over a
billion random amino acid sequences known as nanobodies, that can act as synthetic antibodies, able to bind with target disease antigens. This vast library of nanobodies is then successively screened against the antigen
in question, for example sGP in the case of Ebola. Only those nanobodies
that show strong binding affinity for the antigen are used for sensing.

Next, the selected nanobodies are affixed to gold nanoparticles that
will act as probes to identify and bind with antigens present in a
blood sample. In the study, a pair of two high affinity nanobodies were selected and attached to gold nanoparticles. This approach improves both
the sensitivity and specificity of the test.

The key innovation of Nano2RED is the way the antigen detection is
registered.

As disease antigens in the sample are recognized by the nanobodies,
they bind together, forming clusters of bound nanobody and antigen, like islands of algae floating on the sea surface. "Basically, an antigen
works like superglue to bring the nanoparticle together," Wang says.

A "golden" opportunity in the fight against infectious disease The
gold nanoparticles provide a stable platform to hold the nanobodies in
place. Once enough binding has taken place, the bound clusters begin to
sink to the bottom of the vessel. This can be detected with the naked eye
in the form of a color change. The solution becomes lighter in color as
the gold- nanoparticle-carried antigen-antibody clusters precipitate out, signaling detection of the pathogen.

While full precipitation of nanoparticle clusters leading to a test
result can normally take several hours, the process can be sped
up by centrifuging the sample, which eliminates the wait time for precipitation. In this case, just 15-20 minutes are sufficient for a
result. "Of course, the gold nanoparticles are heavy, and that helps
quick sedimentation, too." Wang added.

Gold nanoparticles also work to display color, not yellow but red here,
by absorbing light from a narrow spectral range. This absorbance feature allowed the Wang lab to invent a tiny, inexpensive device that converts
this color change into an electrical signal, using color-matching
semiconductor LEDs and photodetectors. Such instruments produce a rapid
and accurate readout of assay results, whose limits of detection are
comparable to or better than costly lab- based spectroscopy methods.

The test also delivers quantitative results based on amount of antigen detected. This could be vitally useful for estimating disease severity as
well as time elapsed since the infection event. In the future, the test
results can be digitized by circuits and conveniently transmitted via
internet to anywhere in the world for data analysis and further scrutiny,
which could be important to government policy decision-making processes
and timely interruption of the transmission.

Nano2RED requires only a tiny blood sample, typically around 20
microliters.

"This also makes gold inexpensive in our case, because the mount we need
is so tiny." Wang said. Unlike conventional methods, Nano2RED is also very simple to use with minimal training involved for healthcare personnel. It
does not require any time-consuming and expensive incubation, washing, fluorescent labeling or amplification.

Future research will help improve the assay's limits of detection even
further and modify its detection capacities to include virion particles, extracellular vesicles, small molecules, and nucleic acids. "There is
certainly still a lot to explore" Wang added, "but we so far have a
happy marriage between engineering disciplines and biology. And that is
what we will continue to work on." This research was supported by the
National Science Foundation and National Institutes of Health.

========================================================================== Story Source: Materials provided by Arizona_State_University. Original
written by Richard Harth. Note: Content may be edited for style and
length.


========================================================================== Journal Reference:
1. Xiahui Chen, Shoukai Kang, Md Ashif Ikbal, Zhi Zhao, Yuxin Pan,
Jiawei
Zuo, Liangcai Gu, Chao Wang. Synthetic nanobody-functionalized
nanoparticles for accelerated development of rapid, accessible
detection of viral antigens. Biosensors and Bioelectronics, 2022;
202: 113971 DOI: 10.1016/j.bios.2022.113971 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/02/220202111830.htm

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