Biological Microchips for Medical Diagnostics

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Biological Microchips forMedical Diagnostics
Dmitry A. Gryadunov
Engelhardt Institute of Molecular Biology Russian
Academy of Sciences
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Gel-based Biological Microchips
Academician
Andrei Mirzabekov 1937 - 2003
Director Engelhardt Institute of Molecular
Biology
Founder of the technology
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Gel-based Biochip Technology
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Advantages 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.
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Principle of biochip operation
Light
Chamber Cover
Fluorescently labeled sample molecules
Sample input
Biochip elements with immobilized probe molecules

Image of the biochip elements
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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 ??
022?2006/3777-06)
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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
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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
  hereditary 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
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Biochip technology for analysis of infectious
agents
Development of Techniques Development of Custom
Biochips

• Biochips for TB analysis
• First-line (RIFINH) drug resistance
• Second-line (FQ) drug resistance
• Spoligotyping
• Species identification
• Blood-Borne Pathogens
• Food-Borne Bacteria
• Emerging Diseases
• Orthopoxviruses, Herpesviruses
• Influenza A virus subtyping
• Hepatitis C subtyping
• Resistance in HIV-1
• Neonatal Infections

• Optimization of Hybridization
• On-Chip PCR Amplification
• (including allele-specific)
• On-Chip Real-time PCR
• Cartridges for Automatic sample processing and
  nucleic acids isolation
• Lab-on-a-Chip Development

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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
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Principal Scheme of the Point Mutation
Detection by Hybridization on Biochip
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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
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This product is hybridized to the on-chip
immobilized probes specific to both wild-type and
mutant DNA
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NNNNNNNN A NNNNNNNN
A
N N
NNNNNNNN A NNNNNNNN
NNNNNNN NNNNNNN
NNNNNNNN G NNNNNNNN
NN NNNNNNNN T NNNNNNNN
The fluorescence intensities are compared to find
out whether tested DNA bears a mutation or not
Perfect duplex
Imperfect duplex
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Identification of M. tuberculosis drug resistance
Wild Type
Multidrug-resistant TB
His 526gtTyr (rpoB)
No signal
Ser 315gtThr (katG)
No signal
IS 6110 probe
IS 6110 probe
Routine therapy
Second line drugs should be used
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WorldWide Application of Gel-based Biochip
Technology

• 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

• Institute of Phtisiopulmonology
• BioGlot company (St-Petersburg)

Center National de Genotypage (Evry, France)
Laboratoire de Virologie (Toulouse, France)
State Research Center VECTOR, Novosibirsk
George Washington University (Seattle,USA)
Scientific Research Institute for TB, Novosibirsk
Center for Biologic Evaluation Research
FDA(Rockville, USA)
Kemerovo Anti-TB Health Center
Krasnoyarsk Anti-TB Health Center
University of Arkansas for Medical Sciences
(Little Rock, USA)
General Optics Engineering (Seul, South Korea)
Argonne National Laboratory (Chicago, USA)
National Center for Cardiology and Internal
Medicine, Bishkek, Kyrgyzstan
Saratov Anti-TB Health Center Kazan Anti-TB
Health Center
Rostov Anti-TB Health Center
Center for Disease Control (Atlanta, USA)

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

The Maternal Infantile Institute (IMIP), (Recife,
Brazil)
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TB-Biochip-2 for identification of
fluoroquinolone-resistant M. tuberculosis strains
TB-Biochip-2 identifies mutations in QRDR region
of DNA gyrase gene (gyrA)

• Identifies
• More than 80 of FQ-resistant
• strains

Sensitivity 93 Specificity 100 (based on
analysis gt3000 sputum samples)
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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 ?? 03262004/0889-04
TB-Biochip-2
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 ?? 01012006/3527-06
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Real-time amplification of nucleic acids on the
biochips
Initial amplification stage
Exponential amplification

• Annealing of DNA template and
• extension of the immobilized primers

b) Annealing and extension of the solution
primers
?) On-chip amplification
product
Fluorescence dye
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Quantitative identification of blood-born
pathogens using multiplex on-chip PCR
Amplification of HIV-1 cDNA
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Simultaneous quantitative identification of
HIV-1, HBV, HCV by the on-chip PCR
A kinetics of simultaneous accumulation of
fluorescence signals in different gel elements
of the biochip
B Analysis of melting temperatures in different
gel elements
? Fluorescence image of the biochip after 22
cycle of amplification
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HCV-Biochip for identification of Genotype and
Subtype of Hepatitis C virus
Engelhardt Institute of Molecular Biology
(Russia)
Laboratory of Virology Toulouse University
Hospital (France)
Providing NS5B region sequences Database for
construction of probes
Development of biochip for HCV genotyping
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

HCV-Biochip

• Identifies
• 6 Genotypes
• 36 Subtyoes

• Specificity of analysis
• 100 for genotype
• gt96 for subtype

Tested in clinical trials in Russia and France
(more than 500 samples were tested)
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Biochip for identification of Influenza A virus
subtypes
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Genotyping of strains belonging to H5N1 influenza
A virus and H1N1 influenza A virus
H5N1
H1N1
Avian Flu H5N1 Strain A/chicken/Novosibirsk/64/0
5 Genotype 2.2. Vs Strain A/chicken/Primorje/1/08
Genotype 2.3.2
Human Flu H1N1 Strain A/Puerto Rico/8/34/Mount
Sinai Vs Swine Flu H1N1 Strain
A/California/07/2009
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Diagnostic Kit LeukoGen-Biochip for
determination of chromosomal translocations in
human genome
Registration certificate No ?? 012?2005/2668-06
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 Specific therapy with STI571 Bone
marrow transplantation
Acute promyelocytic leukemia The prognosis is
favorable Specific therapy with all-trans
retinoic acid drugs.
Acute lymphoblastic leukemia The prognosis is
bad High-dose chemotherapy
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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
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BIOCHIPS is an efficient platform for
Single-nucleotide polymorphism analysis
Presymptomatic Diagnostics
Identification of personality
Pharmacogenetics
Preventive Medicine
Analysis of multifactorial diseases (Cancers,
cardiovascular diseases, etc)
GENETIC PASSPORT
Nasedkina TV et al.. Molecular Diagnostics
Therapy. 2009 Vol 13(2)91-102.
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Protein Biochips for quantitative analysis of
tumor markers OM-Biochip, OM-Biochip (PSA)
Protein biochip in transmitted light
(i)
1 2 3 4 5 6 7 8 9

• immobilized antibodies
• for total PSA form (PSA-36)

(ii)

• immobilized antibodies
• for free PSA form (PSA-30)

Fluorescence image of the biochip after analysis
(iii)
- PSA antigen
Registration certificates No ??? 2007/01418 No ??
? 2008/03415
Quantitative analysis
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Joint French-Russian Laboratory in
high-throughput, high-volume and high-resolution
Proteomics
Engelhardt Institute of Molecular
Biology (Moscow, Russia)
Centre National De Genotypage (Evri, France)
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 ?-1
• ?-reactive protein
• - Prostate-specific antigene
• - Myoglobin
• - Calcitonin

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