BIOCHIP-IMB 59_ - Engelhardt Institute of Molecular Biology by pptfiles


									Biological Microchips for Medical Diagnostics
Dmitry A. Gryadunov
Engelhardt Institute of Molecular Biology

Russian Academy of Sciences

Gel-based Biological Microchips

Academician Andrei Mirzabekov
1937 - 2003
Director Engelhardt Institute of

Molecular Biology Founder of the technology

Gel-based Biochip® Technology

specimen molecules probe molecules

3D gel pad

Glass surface (2D)

of 3-dimensional BIOCHIPS

Immobilization capacity is 1000x higher for 3D biochips which increases analysis sensitivity. There is no contact between immobilized probe molecules and support. Immobilized molecules retain their biological activity. Each cell acts as a nanotube wherein any reaction can be performed.

High specificity of the probes and strong signals provide for very good discrimination level. Diagnostics can be done with simple and non-expensive equipment.

Principle of biochip operation

Chamber Cover

Fluorescently labeled sample molecules

Sample input

Biochip elements with immobilized probe molecules
Image of the biochip elements

Biochip Analyzer

Universal for any type of biochips produced by EIMB High speed and Sensitivity User-friendly software for medical personnel Certified in clinical trials (No ФС 022а2006/3777-06)

Biochips Production Facilities
Biochips manufacture is currently certified according to GMP and ISO 13485 standards

Complex for transferring of biomolecules to wafers to produce matrices with nanotubes

Currently biochips production facilities are able to manufacture more than 400 000 biochips per year
Automatic quality control unit

Applications of the Biochips
Analysis of microorganisms and viruses including:  Identification and Species Differentiation  Analysis of genes and mutations responsible for pathogenicity and drug resistance  Genotyping Analysis of human genome including:  Identification of chromosomal translocations responsible for different types of leukemia

 Identification of SNP responsible for predisposition to cancer, cardiovascular and hered diseases  Identification of personality (Forensic studies) Protein biochips:  Quantitative analysis of tumor markers  Integration of Mass-spectrometry and biochips for quantitative parallel identification of proteins in clinical samples Studies of the thermodynamics and specificity of DNA-DNA, protein-protein, and DNA-protein interactions

Biochip technology for analysis of infectious agents
Development of Techniques:
Optimization of Hybridization On-Chip PCR Amplification (including allele-specific) On-Chip Real-time PCR Cartridges for Automatic sample processing and

Development of Custom Biochips:
Biochips for TB analysis: First-line (RIF&INH) drug resistance Second-line (FQ) drug resistance Spoligotyping Species identification

nucleic acids isolation
Lab-on-a-Chip Development

Blood-Borne Pathogens Food-Borne Bacteria Emerging Diseases

Orthopoxviruses, Herpesviruses
Influenza A virus subtyping Hepatitis C subtyping Resistance in HIV-1

Neonatal Infections

Analysis of mycobacteria genomes using biochips
 Identification of mutations responsible for drug resistance* - first-line drugs (rifampin and isoniazid) (TB-biochip (Rif), TB-biochip (MDR)) - second-line drugs/ fluoroquinolones (TB-biochip-2)
 Genotyping of strains belonging to mycobacterium tuberculosis complex (spoligotyping)  Mycobacteria species differentiation
* Certified by Russian Ministry of Health

Principal Scheme of the Point Mutation Detection by Hybridization on Biochip
Fluorescent labeling of a tested DNA is performed using asymmetrical PCR with a labeled primer or labeled dNTPs
As a result, the sample becomes enriched in a single-stranded labeled product This product is hybridized to the onchip immobilized probes specific to both wild-type and mutant DNA


The fluorescence intensities are compared to find out whether tested DNA bears a mutation or not



Imperfect duplex

Perfect duplex

Identification of M. tuberculosis drug resistance
Wild Type
No signal

Multidrug-resistant TB
His 526>Tyr (rpoB)

No signal

Ser 315>Thr (katG)

IS 6110 probe

IS 6110 probe

Routine therapy

Second line drugs should be used

WorldWide Application of Gel-based Biochip Technology
• Institute of Phtisiopulmonology • • “BioGlot” company (St-Petersburg) • Center National de Genotypage (Evry, France) Laboratoire de Virologie (Toulouse, France) George Washington University (Seattle,USA) Center for Biologic Evaluation & Research FDA(Rockville, USA) University of Arkansas for Medical Sciences (Little Rock, USA) Argonne National Laboratory (Chicago, USA) Center for Disease Control (Atlanta, USA) The Maternal Infantile Institute (IMIP), (Recife, Brazil)
Saratov Anti-TB Health Center Kazan Anti-TB Health Center • • • • Moscow Scientific Anti-Tuberculosis Center Central Research Institute for Tuberculosis (Moscow) Institute of Phtisiopulmonology (Moscow) Institite of plant physiology (Moscow) Moscow Prison „Matrosskaya tishina‟ State Research Center for Applied Microbiology, Obolensk

State Research Center VECTOR, Novosibirsk Scientific Research Institute for TB, Novosibirsk Kemerovo Anti-TB Health Center Krasnoyarsk Anti-TB Health Center General Optics Engineering (Seul, South Korea) National Center for Cardiology and Internal Medicine, Bishkek, Rostov Anti-TB Kyrgyzstan Health Center

• Ural Institute of phtisiopulmonology • Ekaterinburg Anti-TB Health Center

for identification of fluoroquinolone-resistant M. tuberculosis strains
TB-Biochip-2 identifies mutations in QRDR region of DNA gyrase gene (gyrA)
More than 80% of FQ-resistant strains

Sensitivity 93% Specificity 100% (based on analysis >3000 sputum samples)

TB – Biochip
Simultaneously Identifies: about 95% of Rifampin-resistant TB strains more than 80% of Isoniazid-resistant TB strains 4 year of successful application in Russia and abroad Validated in Centers for Disease Control and Prevention (USA) Currently uses in 20 anti-tuberculosis centers of Russia Tested with more than 10000 clinical samples (sputum, lavage, etc.) Registration Certificate of Russian Ministry of Health No ФС 03262004/0889-04

Simultaneously Identifies: more than 85% of Fluoroquinolone-resistant TB strains Currently uses in 6 anti-tuberculosis centers of Russia Tested with more than 3000 clinical samples Registration Certificate of Russian Ministry of Health No ФС 01012006/3527-06

Real-time amplification of nucleic acids on the biochips

t oC n

Initial amplification stage: a) Annealing of DNA template and extension of the immobilized primers

Exponential amplification:
b) Annealing and extension of the solution primers с) On-chip amplification product

Fluorescence dye

Quantitative identification of blood-born pathogens using multiplex on-chip PCR

Amplification of HIV-1 cDNA

Simultaneous quantitative identification of HIV-1, HBV, HCV by the on-chip PCR

Normalized fluorescence signal

Cycle No A – kinetics of simultaneous accumulation of fluorescence signals in different gel elements of the biochip B – Analysis of melting temperatures in different gel elements С – Fluorescence image of the biochip after 22 cycle of amplification

HCV-Biochip for identification of Genotype and Subtype of Hepatitis C virus
Engelhardt Institute of Molecular Biology (Russia)
Development of biochip for HCV genotyping

Laboratory of Virology Toulouse University Hospital (France)
Providing NS5B region sequences Database for construction of probes Performing evaluation of the developed approach using collection of clinical samples with HCV (more than 2000 samples)

The objective of the kit

 Determination of duration and doze for treatment  Prognosis (acute/chronic cirrhosis/liver cancer)  Identification of most virulent and drug-resistant types  Epidemiological studies

Identifies: 6 Genotypes 36 Subtyoes Specificity of analysis: 100% for genotype >96% for subtype
Tested in clinical trials in Russia and France (more than 500 samples were tested)

Biochip for identification of Influenza A virus subtypes

Virus A/WSN/33



A/FPV/Rostock/34 A/Chicken/Novosibirsk/2005

H7N1 H5N1

Genotyping of strains belonging to H5N1 influenza A virus and H1N1 influenza A virus H5N1 H1N1

‘Avian Flu’ H5N1


‘Human Flu’ H1N1 ‘Swine Flu’ H1N1


Strain A/chicken/Novosibirsk/64/05 Genotype 2.2. Vs Strain A/chicken/Primorje/1/08 Genotype 2.3.2

Strain A/Puerto Rico/8/34/Mount Sinai Vs Strain A/California/07/2009

Diagnostic Kit ‘LeukoGen-Biochip’ for determination of chromosomal translocations in human genome
Registration certificate No ФС 012а2005/2668-06

An identification of chromosomal translocations allows to establish the precise diagnosis, to predict the severity of disease and to prescribe the adequate therapy

Chronic myeloid leukosis
The prognosis is unfavorable

Acute promyelocytic


Acute lymphoblastic leukemia
The prognosis is bad High-dose chemotherapy

Control Gene ABL

The prognosis is favorable Specific therapy with all-trans retinoic acid drugs.

Specific therapy with STI571
Bone marrow transplantation

Identification of Personality in Forensic Studies
victim suspected 2 suspected 3

suspected 1

suspected 4

EIMB& Forensic Science Center, Ministry of the Interior of Russia

Material from the place of crime

BIOCHIPS is an efficient platform for Single-nucleotide polymorphism analysis

Presymptomatic Diagnostics Preventive Medicine Analysis of multifactorial diseases (Cancers, cardiovascular diseases, etc)


Identification of personality

Nasedkina TV et al.. Molecular Diagnostics & Therapy.
2009 Vol 13(2):91-102.

Protein Biochips for quantitative analysis of tumor markers OM-Biochip, OM-Biochip (PSA)
Protein biochip in transmitted light












- immobilized antibodies for total PSA form (PSA-36) - immobilized antibodies for free PSA form (PSA-30)

Fluorescence image of the biochip after analysis


- PSA antigen

Quantitative analysis

Registration certificates: No ФСР 2007/01418 No ФСР 2008/03415

Joint French-Russian Laboratory in high-throughput, high-volume and high-resolution Proteomics
Centre National De Genotypage (Evri, France) Engelhardt Institute of Molecular Biology (Moscow, Russia)

An objective: The integration of biological microchips technology and mass-spectrometry (MALDI TOF MS) for multiple parallel quantitative detection of proteins and analysis of protein-protein and protein-ligand interactions in proteomics
Quantitative MALDI MS assay of proteins involves tagging of proteomes with mass tags, interaction of proteomes with biochips with immobilized antibodies, on-chip digestion with proteases, MALDI MS assay The developed approach was applied for quantitative analysis of proteins in blood serum : - apolipoprotein А-1 - С-reactive protein - Prostate-specific antigene - Myoglobin - Calcitonin

Analysis of apolipoprotein in blood serum

From Gel-based biochips to Integrated Lab-On-a-Chip Solution for complete analysis of sample
Sample injection Processing (lysis) NA Extraction PCR Hybridization on the biochips Detection

Current : Sample processing, detection, and data analysis are all separated and require lab conditions, skilled personnel, significant reagents volume, and expensive equipment to perform the analysis Proposed: Integrated platform includes all elements. It results in increased accuracy and sensitivity; reduced detection time, reagents volume and overall analysis cost; and finally substitute complex lab work with user - friendly field device
Module for sample processing and NA isolaton
Mixing of reagents for PCR and isolated NA Real-time On-chip PCR

Qualitative / Quantitative Analysis

Sample Injection


Scheme of automated NA isolation using disposable cartridge

Prototype of the integrated platform with cartridges for automated sample processing and nucleic acids isolation

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