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).
STRUCTURE AND OPERATION
This system consists of a lab-on-a-chip where our sample is a nasopharyngeal swab in saline
solution. This sample is deposited in the sample spot and let the sample go over all the microfluidic
length. Finally, the sample ends in a spot where we previously set our molecule quantum
dot-antibody(QDs-Ab) solution and the two solutions mix, QDs-Ab bound with the SARS-CoV-2 spike
protein will be excited with a UV led and the photoreceptor will capture the fluorescence and analyze
the sample in order to deliver a COVID positive or negative results (figure 1). In addition, for greater
sensitivity, we will add a second microfluidic channel and receive two different signals (also
corresponding calibration curve), these can be compared through calculations of ΔI.
Figure 6. Diagram of COVID-19 lab-on-a-chip device procedure. A sample is deposited in the sample
spot and goes over all the microfluidic length. Finally, the sample ends in a spot with QDs-Ab solution
and the two solutions mix to finally be detected by a photobiosensor.
TRANSDUCTOR
The sensor for measuring the intensity of the light emitted by the reaction is a light-dependent resistor
(LDR) that generates an output signal in the form of variations in the electrical current flowing through
the component that can be calibrated to perform an estimation of the presence of SARS-CoV-2 on
someone sample.
The intensity of light is inversely proportional to electrical resistance and this change can be calibrated
in an electronic circuit to estimate the amount of virus on the sample.
Figure 7. Light-dependent resistor and curve of response.
To produce the expected reaction it is necessary to irradiate the sample with ultraviolet light which is
in the range of 10 to 400 nanometers.
Figure 8. Ultraviolet electromagnetic spectrum.
OPTOELECTRONIC SYSTEMS ACQUIRE DATA.
The part A corresponds to the process of conversion of fluorescence signal to an electrical signal.
QUANTUM DOT SIGNAL
The variation of photoluminescence (PL) spectra in CdSe/ZnS quantum dots (QDs) at the conjugation
to biomolecules, in this case, two types of CdSe/ZnS QDs with different CdSe core sizes (5.4 and 6.4
nm) and emissions (605 and 655 nm) were studied before and after the conjugation to
anti–Interleukin-10 (IL-10) and anti-Pseudo rabies virus (PRV) ABs. PL spectra varies essentially in
bioconjugated QDs: the PL intensity decreases on 10–50% and the PL high energy spectral shift
appears (Figures 8. 1b, c,and 9b, c). Simultaneously, the full width at half maximum (FWHM) of PL
bands increases, and the PL band shape becomes asymmetric with essential high energy tails
(Figures 8. 1b, c and 9b, c). [13]
The outcome we are expecting in case of a positive diagnosis for SARS-CoV 2 is a decrease in the
photoluminescence intensity as described before.
Figure 8. PL spectra of three non-conjugated
605N (a) and three bio-conjugated 605-PRV
(b) and 605-IL-10 (c) QD ensembles.[13]
Figure 9. PL spectra of three non-conjugated
655N (a) and three bio-conjugated 655-PRV
(b) and 655-IL-10 (c) QD ensemble.[13]
VIRUS CONCENTRATION ESTIMATE
Research by Henan University carried out practical field samples using sixty human throat swab
samples where QDs-LFIA and real-time PCR were compared and it shows that all positive samples
with low real-time PCR were detected by QDs- LFIAS with high accuracy. A matter of fact, compared
with real-time PCR, the QDs-LFIAS had an accuracy of 95%, while that of the commercial influenza A
antigen rapid diagnostic test kit (colloidal gold) was 56.7%.
Figure 10. Results of the research by Henan University “
DLS data of QDs and conjugated QDs-Ab.
(a) Carboxyl-functionalized QDs. (b) Antibody conjugated QDs. The average hydrodynamic size of
CdSe/ZnS QDs was 42.86 nm and this size increased to 109.5 nm after conjugation with antibodies.
(c) Fluorescence intensity vs concentration graphic” [11].
|
Figure 11. (A) Specificity tests of QDs-LFIAS. (B) Fluorescence intensity scans at different
concentrations of influenza A virus subtypes. Shows QDs-LFIAS could detect all the subtypes of
influenza A virus used but none of the other type antigens. B shows QDs-LFIAS could detect the
subtypes of influenza A virus with high sensitivity.
Due to this information, an estimate of the relationship of the intensity of fluorescence with the
concentration of the virus SARS-COV2 could be made.
ADVANTAGES AND DISADVANTAGES
Table 1. Advantages and disadvantages of Quantum Dot-Based biosensor for rapid detection of
SARS-CoV-2.
Advantages
Disadvantages
Rapid test: It takes a few minutes to give the
result.
Risk of error due to misuse of the test.
High sensitivity because it can read a small virus
concentration
Cost depends on the chemical materials and the
kind of narrow-band filter used.
Low-cost biosensor
High specificity is given by the antibody and
recognition element.
CONCLUSION
Through this research, we develop an idea of an ultrasensitive, rapid and low-cost lateral flow immune
sensor for SARS-COV2. A QDs-LFIAS method, which rapidly analyzed the sample through one step.
We estimate that QD-LFIAS could detect spike protein antibodies with high sensitivity and specificity.
This was more sensitive than that of traditional point-of-care testing methods. The specificity and
reproducibility were shown to be good. Owing to previous studies we know that real patient samples
demonstrate that the QDs-LFIAS had higher accuracy, and detection of nasal-pharyngeal swab
samples makes it more rapid and efficient for identification of viral infection and improves patients
management.
TEAM
This joint project is the product of two weeks of intensive studying different scientific areas, innovation
and entrepreneurship and a multidisciplinary team where each member of the team focused on the
area of the biosensor corresponding to this area. Besides that, It was highly relevant to count on the
collaboration of other members of the task teams to complement our information and make the
monitoring of the innovation track in the diagnosis more complete. Furthermore, we want to express
our gratitude to our assigned instructor Siddharth Doshi, for his understanding, comments and
suggestions to improve this project. We also sincerely thank Silvia Lorena Montes Fonseca from task
1 for providing us the scientific knowledge and practical skills which will help us to understand and fix
many of the problems we ran into. And last but not least, highlight the participation of José Luis Tlaloc
Sauceda, member of our team who supported members of Challenge CdCMx in the development of
web pages, putting an extra effort in this project.
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