Quantum Dot-Based biosensor for rapid detection of SARS-CoV-2
María José Vásquez Juárez - Instituto Tecnológico y de estudios superiores de occidente
Daniella María Joselyn Hernández Pérez - Instituto Politécnico Nacional de México
Naomi Martinez Ayala - Instituto Politécnico Nacional de México
Luis Tlaloc Sauceda Alvarez - Universidad de Guadalajara
Clubes de ciencia challenge 2020, 15/08/2020.
ABSTRACT
Rapid, specific, sensitive, low cost and easy to use SARS-CoV-2 tests for use in emergency and
critical point of a pandemic for which diagnostics are essential, as well as for application in low
resource settings. This document shows a new biosensor to perform rapid tests that serve the
diagnosis of SARS-CoV-2 is using a CdSe/ZnS quantum dot (QDs) conjugated with specific
antibodies and used as fluorescent labels. The antibodies used for detection are specific for the spike
protein and ensure the accuracy and specificity. The biosensor incorporates a deposit for the sample
(nasopharyngeal exudate fluid in saline solution) which pass through a microfluidic channel
(dispersion) ending in a deposit containing a solution of QDs-Ab that emit a signal being excited by
ultraviolet light, this signal emitted by the QDs will be captured by a photoreceptor (transducer) which
obtain a spectral difference used for the diagnosis.
INTRODUCTION
Coronavirus disease 2019 (COVID-19), formerly known as 2019-nCoV acute respiratory disease, is
an infectious disease caused by SARS-CoV-2, a virus closely related to the SARS virus. The disease
is the cause of the 2019–20 coronavirus outbreak. The structure of 2019-nCoV consists of the
following: a Spike protein (S), hemagglutinin-esterase dimer (HE), a membrane glycoprotein (M), an
envelope protein (E) a nucleocapsid protein (N) and RNA. Coronavirus invades cells through Spike
(S) glycoproteins, a class I fusion protein. It is the major viral surface protein that coronavirus uses to
bind to the human cell surface receptor. It also mediates the fusion of host and viral cell membrane,
allowing the virus to enter human cells and begin infection. The spike protein is the major target for
neutralizing antibodies and vaccine development.
The protein modeling suggests that there is a strong interaction between the Spike protein
receptor-binding domain and its host receptor angiotensin-converting enzyme 2 (ACE2), which
regulate both the cross-species and human-to-human transmissions of COVID-19. The recent study
has shown that the SARS-CoV-2 spike protein binds ACE2 with a higher affinity than the SARS-CoV
spike protein. [1]
Nanotechnology can be exploited to improve the utility of fluorescent markers used for diagnostic
purposes. The mechanism of imaging is determined by the type of modality used for imaging such as
nanocarriers including liposomes, dendrimers, Buckyballs, and numerous polymers and copolymers.
They can be filled with a large number of imaging particles such as optically active compounds and
radionuclides for detection with imaging equipment. Although fluorescent markers are routinely used
in basic research and clinical diagnostic applications, there are several inherent disadvantages with
current techniques, including the requirement of color-matched lasers, the fluorescence bleaching,
and the lack of discriminatory capacity of multiple dyes. Fluorescent nanocrystals potentially
overcome these issues [2]. Quantum dots are crystalline clumps of a few hundred atoms, coated with
an insulating outer shell of a different material [3]. When a photon of visible light hits such a minute
particle, a quantum-physics reflection confines all the photon’s energy to the crystal core before being
emitted as an extraordinary bright fluorescence. The QDs absorb light at a wide range of wavelengths
but emit almost monochromatic light of a wavelength that depends on the size of the crystals [4]. The
visualization properties of quantum dots (fluorescence wavelength) are strongly size-dependent. The
optical properties of quantum dots depend upon their structure as they are composed of an outer shell
and a metallic core. Quantum dot core is usually made up of cadmium selenide, cadmium sulfide, or
cadmium telluride. The outer shell is fabricated on the core with high bandgap energy in order to
provide electrical insulation with the preservation of fluorescence properties of quantum dots. The
fine-tuned core and shells with different sizes and compositions with visualization properties of
specific wavelengths provide a large number of biomarkers [5]. Quantum dots are conjugated with
different ligands in order to obtain specific binding to biological receptors. Quantum dots offer
significant advantages over the conventional dyes such as narrow bandwidth emission, higher
photostability, and extended absorption spectrum for the single excitation source. Moreover, the
challenge of hydrophobicity in quantum dots has been overcome by making them water-soluble. An
example of the aqueous quantum dots with long retention time in biological fluids is the development
of highly fluorescent metal sulfide (MS) quantum dots fabricated with thiol-containing charged groups
[6].
Microfluidics concerns design, fabrication and experiments of miniaturized fluidic systems, which has
undergone rapid developments during the last decade [7]. As an interdisciplinary area, this rapidly
growing field of technology has found numerous applications in biomedical, diagnostics, chemical
analysis, automotive and electronics industries. The sorting of micron-sized objects in a continuous
flow is required for a wide variety of applications, including chemical syntheses, mineral processing
and biological analyses. PDMS is used for the construction of microfluidic devices using lithography
and a mold replication process. The microchannels formed in the PDMS are sealed with glass using a
sealing process. One of the most commonly used techniques to obtain irreversible seals is by
exposing surfaces to oxygen plasma [8].
A light-emitting diode (LED) is a semiconductor light source that emits light when current flows
through it. Electrons in the semiconductor recombine with electron holes, releasing energy in the form
of photons. The color of the light (corresponding to the energy of the photons) is determined by the
energy required for electrons to cross the bandgap of the semiconductor. There are many ways to
measure this energy or its variations if it is a source that changes over time.
A light-dependent resistor (LDR) is an electronic device that changes its electrical resistance with
variations in the light striking its surface, and we can take advantage of these qualities to make
fluorescent reaction measurements.
METHOD
BIOMARKER “QDs”
Figure 1. Diagram of Schlenk sintesis for QDs
core CdSe [9].
Figure 2. Diagram of SILAR technique for QDs
shell Zn [10].
Figure 3. Diagram of the conjugation of spike protein antibodies to QDs via oxidized Fc-carbohydrate
groups [11].
Figure 4. Diagram of PDMS development and design microfluidic platform [12].
RECOGNITION ELEMENT
SARS-COV-2 structure contains four important proteins: envelope, nucleocapsid, matrix and spike.
This last one is highly immunogenic and it is a major transmembrane protein, because of that reason,
it is best suited to be used as a diagnostic antigen. It also exhibits amino acid sequence diversity
exclusive of SARS-COV-2.
Giwan, S., et.al. in 2020 used the SARS-CoV-2 spike antibody immobilized onto a Field-Effect
Transistor-Based Biosensor and they verified the performance of the antibody by enzyme-linked
immunosorbent assay (ELISA). Their results prove that the antibody bound specifically to the
SARS-CoV-2 spike protein, therefore, is suitable in the detection of SARS-CoV-2. [12]
The SARS-Cov 2 Spike S2 monoclonal antibodies were first produced in mice by injecting an
immunogenic fragment of the S2 subunit of SARS-CoV 2 to generate murine monoclonal antibodies,
which are later purified from the cell culture with a protein affinity column. Now they are produced in
in-vitro
rabbit cell cultures. [1]
Figure 5. Antibody binding to coronavirus spike protein. Molecular model of an antibody (blue) binding
to the spike (S) protein (red) of the new coronavirus SARS-CoV-2. Gaernet (2020).