Combatting COVID-19 NANOBIOSENSORS IN FIGHT AGAINST ... · •Diagnostics is the FIRST step in medical care • Follow-up treatments are based on Diagnostics DIAGNOSTICS: today and

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

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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

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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!!!