Combatting COVID-19NANOBIOSENSORS IN FIGHT AGAINST CORONAVIRUS
Prof. Laura M. LechugaNanobiosensors and Bioanalytical Applications group (NanoB2A)
Catalan Institute of Nanoscience and Nanotechnology (ICN2)
CSIC, BIST & CIBER-BBN
Barcelona, España
@nanob2a_groupnanob2a.icn2.cat
• Diagnostics is the FIRST step in medical care• Follow-up treatments are based on Diagnostics
DIAGNOSTICS: today and tomorrow
Abbott’s FreeStyle Libre
Point-of-care (POC) nanobiosensorDrop of sample
Main Goal in Diagnostics
Easy diagnostics High sensitivity and Fast Multiplexing capabilities User-friendly/minimum operation Minimum sample
Pesonalised Treatment
Basic test Specialized tests & sophisticated techniques
Personalized
Diagnostics
Laboratory Techniques
Time consuming
High Sample volume
Trained personnel
Laboratory/clinical
installations
Bulky instrumentation
Nanotechnology is providing highly competitive POC Nanobiosensor devices for Diagnostics
(proteins, DNA, antibodies, cell receptors,..)
(Optical, Electrochemical, Gravimetric, mechanical..)
Biosensors can detect any substance with a high sensitivity using a specific and selectivebiomolecular recognition in real time and very fast
Substances detected: proteins, DNA, pathogens, virus, bacteria, toxic pollutants, chemical andbiological warfare agents (applications almost endless),
NANOBIOSENSOR DEVICE
FAST, DIRECT, LABEL-FREE, HIGH SENSITIVITY, LOW SAMPLE VOLUME
Glucose biosensor Pregnancy test i-Stat blood portable analyzer
Abbott’s FreeStyleLibre
DIAGNOSTICS OF COVID-19
Diagnostics strategiesSARS-CoV-2 virus
DETECTION OF THE VIRUS RNA
GENETIC MATERIAL
(Nucleic acid test)
DETECTION OF THE INTACT VIRUS (Antigen detection test)
❶
❷
DETECTION OF ANTIBODIES (Serological test)
❸Spike Protein (100 copies)
RNA
Nucleocapsid Protein (1000 copies)
Lipid Membrane
COVID-19 Diagnostics
DETECTION OF THE VIRUS RNA
GENETIC MATERIAL ❶
Strategy based on the “Polymerase Chain Reaction” technique (PCR)
Nucleic Acid Amplification Tests (RT-PCR)
Advantages: well-established commercial technique,
high sensitivity, specificity, high scalability to thousands of
detection kits.
Limitations: time consuming (2-5 h), reproducibility,
trained personnel, limited to specialized laboratories,
complex instrumentation, price.
From a few DNA copies, and thanks to iterative cycles of, high yield ofgenetic material can be obtained, detected using fluorescent labels
RNA Extraction Conversion to ADNamplification
ResultsSample collectionlabeling
PCR assay
Time to Result and Lab requirements:Main handicaps for massive population testing
DETECTION OF THE INTACT VIRUS
(Antigen detection test)❷
COVID-19 Diagnostics
Advantages: Rapid test (5-15 min), well-established
technique for other diseases (lateral flow
immunoassay), low cost, massive production, at the
point-of-need.
Limitations: limited sensitivity (false negative for low
viral load), reproducibility issues between batches,
qualitative results (YES/NO) but not the viral load.
Detection of the intact virus through the outer virus
proteins (viral antigens) by using specific antibodies
A Viralgenomicanalysis
B Directvirusdetection
C Serologyassay
Positive ResultNegative ResultSample treatment Test with selective antibodies
Sample collection Sample transfer
IDEAL TEST FOR A MASSIVE COVID-19 DETECTION
BUT……
THERE IS NO SOLUTION AVAILABLE YET
COVID-19 Diagnostics
Advantages: Rapid test (5-15 min), well-established
technique, easy sample extraction, non-infective
sample, low cost, massive production, at the point-of-
need, required selectivity.
Limitations: limited sensitivity (false negative and
false positive), qualitative results (YES/NO) but not
the antibody levels, variability of the immunoresponse
in the population, not indicated for infection diagnosis.
DETECTION OF ANTIBODIES
(SEROLOGICAL TEST)❸
Detection of antibodies produced by the infected person during the
disease (detection of antibodies present in blood/serum samples)
Positive ResultNegative ResultSample treatmentTransfer and Test with selective viral antigens
Sample collection
• Most of the Rapid test for serological analysishave NOT the required level of sensitivity (falsepositive and false negative)
• There is no information yet about the duration and the quality of the immunity
• Immunity Passport is NOT a good idea
INDIRECT DETECTION
DIAGNOSTICS OF COVID-19
SARS-CoV-2 virus DETECTION OF THE VIRUS RNA
GENETIC MATERIAL
DETECTION OF THE INTACT VIRUS (Antigen detection test)
❶
❷
Nucleic Acid Amplification Tests (RT-PCR)
A Viralgenomicanalysis
B Directvirusdetection
C Serologyassay
Easy diagnostics at the Point-of-need High sensitivity and selectivity Fast diagnosis (min) User-friendly Minimum sample treatment
NANOBIOSENSORPOC devices
How Nanotechnology can help in the fight against COVID-19?
Opening the route to:RAPID, SENSITIVE, MASSIVE AND QUANTITATIVE DETECTION
Combatting COVID-19: ADVANCED NANOBIOSENSING PLATFORMSFOR POC GLOBAL DIAGNOSTICS AND SURVEILLANCE
CONVAT PROJECT
A new POC Nanophotonics biosensor platform able to
provide an accurate and fast COVID-19 diagnosis withoutrequiring complex equipment.
Own production of antibodies and proteins
Diagnostics of COVID-19 in human samples and clinical
validation (antigen & RNA label-free detection)
Surveillance of SARS-Cov-2 coronavirus in reservoir
animals samples
NANOPHOTONIC BIOSENSORS
How it works?A light beam is travelling inside the biosensor.
When the SARS-CoV-2 virus is trapped by the
specific antibodies, there is change of the
properties of the light.
At the exit, the light is analysed. From this data, we
can know if a person is infected (YES/NO) and if
infected, also the viral load (QUANTIFICATION)
Well demonstrated NANOBIOSENSOR for the diagnostics of other infectious diseases
3 nm AFM
20 biosensorsper chip
1 biosensor
Nanotechnological device
Light interacting with the biomolecules at nm scale
100-200 nm
E. coli
Nanobiosensor
LoD: 4 cfu.mL-1
Fast identification and quantification of bacteria (20 min.)
Genomic detection of the antibiotic resistant profile (30 min.)
250 µL
150 µL orina
Genomic analysis
Single DNA cancer mutations
DNA Epigenetics
microRNAs biomarkers
Messenger RNA
Alternative splicing RNA
Well demonstrated NANOBIOSENSOR for the diagnostics of other diseases
A Viralgenomicanalysis
B Directvirusdetection
C Serologyassay
PROJECT
Specific antibodies for the capture of
complete SARS-CoV-2 virus
Real-time rapid diagnosis
Quantification of the viral load in the
sample.
complementary DNA probes to hybridize
exclusive sequences of the SARS-CoV-2
RNA.
Different DNA probes for similar virus to
identify the presence of other viral species
in the sample.
Detection of antibodies in patients for
serological testing using antigens of SARS-
CoV-2.
Identify asymptomatic individuals,
patients with mild symptoms
Useful for epidemiological studies.
Direct Virus Detection
A Viralgenomicanalysis
B Directvirusdetection
C SerologyassayEvaluation of affinity and specificityAntibody selection
LOD = 46 FFU/mL(R2 = 0.9971)
ICN2 BSL2 facilities
Preliminary results
Pseudotype virus (non-pathogenic)
Reagents: Ag & Ab produced by one Project partner (Monoclonal Abs
and several nanobodies)
Evaluation of Deactivated (UV and thermal) SARS-CoV-2 virus
Real samples from COVID-19 patients at Italian hospital
UV inactivated
SARS-CoV-2/TEM
Thermal inactivated
A Viralgenomicanalysis
B Directvirusdetection
C Serologyassay
0 500 1000
0.0
0.5
1.0
1.5
2.0
2.5
N1 gene (nM)
% S
en
sor
Sig
na
l
0 50 100
0.0
0.5
1.0
1.5
E gene (nM)
%S
ens
or
Sig
nal
LOD = 1 nM LOD = 3 nM
Direct RNA Detection
Sequences selected from WHO recommendation for SARS-CoV-2 PCR detection
• 3 candidate sequences showed 100% similarity to SARS-CoV-2 and 0% matches
to other genomes (E gen CUB, N1 CDC, N gene CUB)
• Exclusion was mainly based on a match with a possible human pathogen (or
related) in nucleotide database.
DNA TARGET DNA TARGET
NO NEED OF PCR AMPLIFICATION
Evaluation of RNA synthetic targets
Viral SARS-CoV-2 RNA supplied by one Project partner
Multiplexed analysis with other coronavirus sequences
Serological tests
A Viralgenomicanalysis
B Directvirusdetection
C Serologyassay
Preliminary results
LOD = 10.1 ng/mL
LOD = 22.2 ng/mL LOD = 13.7 ng/mL
S1 protein
RBD proteinN protein
Evaluation in spike Serum samples
Evaluation of Serum samples from COVID-19
patients & healthy subjects (provided by Vall
D´Hebron Hospital)
Tech transfer to one company in negotiations
ICN2 BSL2 facilities
Summary of CoNVat approach for diagnostics and surveillance of Covid-2019
Point-of-care diagnostics platforms for descentralized analysis
• Point-of-care nanobiosensors are required for fast,
direct, label-free, high sensitivity, low sample volume
and massive diagnostics
• Nanohotonic biosensors are one of the most
competitive technology
• Sensors need to be disposable, that means cheap,
therefore mass production is required
NANOTECHNOLOGY CAN PROVIDE:
Nanob2a.icn2.cat
@NanoB2A_group
THANK YOU!!!GRACIAS!!!
