new biophotonic method and nanostructure sensor instrument ... · robert a. freitas jr.,...

Kharkiv National University of Radio Electronics, UkraineLaboratory of Analytical Optochemotronics

61166, Ukraine, Kharkiv, Lenin Av. 14, phone +38(057)70-20-369, e-mail: [email protected]

Doctor of Physical-Mathematical Science, Professor Mykola

Rozhitskii,Senior Scientific researcher, PhD Dmytro Snizhko,

Scientific researcher Alena Galaichenko

Lyon, FranceNovember 26-28, 2008

Nouvelles

perspectives dans

le domaine

des maladies infectieuses

New Biophotonic

Method and Nanostructure Sensor Instrument for Diagnostis

of Tuberculosis Active Form


One third of Earth population is infected by the latent form of tuberculosis and lives with risk of transition of this illness in the open form. Annually 2 million persons die from tuberculosis.

4

Country 2000 2001 2002 2003 2004 2005 2006Albania 604 555 594 543 547 506 469Andorra 12 10 5 10 7 10 12Armenia 1,333 1,389 1,433 1,538 1,660 2,206 1,767Austria 1,185 1,013 1,044 946 895 928 855Azerbaijan 5,187 4,898 5,142 3,840 5,404 6,034 5,705Belarus 6,799 5,505 5,139 5,106 5,443 5,308 5,142Belgium 1,278 1,321 1,211 1,030 1,128 1,076 1,043Bosnia and Herzegovina 2,476 2,469 1,691 1,740 2,353 2,111 1,778Bulgaria 3,349 3,862 3,335 3,069 3,025 3,225 3,136Croatia 1,630 1,376 1,443 1,356 1,170 1,050 1,029Cyprus 33 40 20 35 30 34 36Czech Republic 1,414 1,291 1,156 1,101 1,027 973 941Denmark 587 494 403 378 356 395 341Estonia 791 708 620 557 537 479 422Finland 527 460 449 392 319 339 280France 6,122 5,814 5,709 5,740 5,004 4,887 4,817Georgia 4,397 4,006 4,490 4,212 4,011 4,501 4,554Germany 9,064 6,959 6,931 6,526 6,007 5,539 5,021Greece 703 503 570 571 668 626 580Hungary 3,073 2,923 2,720 2,507 2,251 1,808 1,687Iceland 13 12 8 5 11 10 13Ireland 386 393 375 354 380 387 416Israel 557 546 485 505 497 402 384Italy 3,501 4,287 3,925 4,234 3,968 3,828 4,145Kazakhstan 25,843 26,224 27,546 26,936 26,493 25,739 23,728Kyrgyzstan 6,205 6,654 6,613 6,172 6,104 6,329 6,174Latvia 1,982 2,000 1,803 1,686 1,579 1,409 1,290Lithuania 2,657 2,598 2,414 2,586 2,036 2,114 2,365Luxembourg 44 31 31 54 31 37 33Malta 16 16 24 6 18 21 30Monaco 0 0 0 n/a n/a n/a n/aNetherlands 1,244 1,408 1,355 1,282 1,316 1,127 1,002Norway 221 276 243 320 278 269 276Poland 10,931 10,153 10,069 9,677 8,698 8,203 8,017Portugal 4,227 4,320 4,381 3,861 3,600 3,303 3,218Republic of Moldova 2,935 3,608 3,769 3,619 4,806 5,141 4,990

Infection of people in the world

Country 2000 2001 2002 2003 2004 2005 2006Romania 27,470 28,580 29,752 28,335 28,570 26,104 24,295Russian Federation 140,677 132,477 128,873 124,041 121,426 127,930 124,689San Marino 1 0 1 1 0 n/a n/aSlovakia 1,010 986 975 904 664 710 673Slovenia 368 359 338 275 249 269 207Spain 7,993 6,851 7,283 7,343 6,015 7,281 7,815Sweden 417 394 375 386 416 539 489Switzerland 544 539 591 554 528 508 461Tajikistan 2,779 3,508 4,052 4,260 4,529 5,460 5,362The former Yugoslav Republic of Macedonia 641 648 686 653 644 598 561

Turkey 18,038 17,263 18,043 17,923 17,543 19,744 19,629Turkmenistan 4,038 3,948 3,671 3,771 3,382 3,191 3,223Ukraine 32,945 36,784 40,175 37,043 38,403 39,608 41,265United Kingdom of Great Britain and Northern Ireland 6,220 6,027 6,889 6,400 7,039 8,173 8,157

Uzbekistan 15,750 17,391 20,588 20,700 20,289 21,513 23,900

0-999

1000-9999

100 000-999 999

1 000 000 and more

Not determine

10 000-99 999

5

Mycobacterium growing on Löwenstein-Jensen

slants of Mycobacterium tuberculosis

Tuberculin skin test

Electron microscopy of Mycobacterium tuberculosis growing in culture (Courtesy

of M. Rohde -M. Singh)

Ziehl-Neelsen staining of Mycobacterium tuberculosis growing

in culture at 1000x magnification


http://who.int


In the world for tuberculosis testing about billion of dollars are spent, and for medicaments treatment of this illness about 300 million dollars are spent annually. Doctor Mario Ravilone (the Director of WHO

department for

tuberculosis prevention): «New, safe and inexpensive diagnostic systems are urgently necessary for simplification of revealing of tuberculosis cases for the world. Most of tuberculosis patient in the world have access only to usual microscopical researches. These methods demand repeated testing, they are capable to "pass" to half of cases of disease, they are ineffective for the patients in parallel infected with a HIV».

10

P. Feynman, "There's Plenty of Room at the Bottom," Engineering and Science (California Institute of Technology), February 1960, pp.22- 36.N. Taniguchi, "On the Basic Concept of 'Nano-Technology', Proc. Intl. Conf. Prod. Eng. Tokyo, Part II, 1974, Japan Society of Precision Engineering.

Richard Feynman

(1918 - 1988)

The The firstfirst

mention of methods which will be named mention of methods which will be named subsequently subsequently nanotechnologynanotechnology::

1959 1959 The Californian Institute of Technology, an The Californian Institute of Technology, an annual meeting of the American Physical Society.annual meeting of the American Physical Society.Richard FeynmanRichard Feynman report report ««ThereThere’’s Plenty of Room s Plenty of Room at the Bottomat the Bottom»»..

Firstly the term «nanotechnology» has usedNorio Taniguchi in 1974. He named with this term He named with this term manufacturing of products with size of manufacturing of products with size of the order of the order of nanometers.nanometers.

Nanotechnology – the area of applied science and the technique, dealing with objects in the size less than 100 nanometersnanometers.


12

Robert A. Freitas Jr.: nanomedicine - tracking, correcting, designing and control over biological systems of the person at molecular level, using developed nanoinstruments and nanostructures.

Nanomedicine combines such unique things, as:– laboratory on chip (lab-on-a-chip);– address delivery of medicines to the damaged cells;– new bactericidal and antiviral agents;– diagnostics of diseases using quantum dots;– nanorobots (nanobots) for repair of damaged cells;– neuroelectronic interface etc.


Possible type of respirocyte

Robert A. Freitas Jr., Nanomedicine, Vol. I: Basic Capabilities, Landes Bioscience, Georgetown, TX, 1999Robert A. Freitas Jr., Nanomedicine, Vol. IIA: Biocompatibility (Landes Bioscience, 2003)Robert A. Freitas Jr., “Exploratory Design in Medical Nanotechnology: A Mechanical Artificial Red Cell,” Artif. Cells, Blood Subst., and Immobil. Biotech. 26(1998):411-430.

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Robert A. Freitas Jr.: the respirocyte is a blood borne 1 micron diameter spherical nanomedical device. The device acts as an artificial mechanical red blood cell. It can deliver 236 times more oxygen to the tissues per unit volume than natural red cells while simultaneously managing carbonic acidity. An individual respirocyte consists of 18 billion precisely arranged structural atoms plus 9 billion temporarily resident molecules when fully loaded. An onboard nanocomputer and numerous chemical and pressure sensors allow the device to exhibit behavior of modest complexity, remotely reprogrammable by the physician via externally applied acoustic signals.


Patrol and detection of a foreign body in vascular system by nanobots

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Source: N

anotech Now

(ww

w.nanotech-now

.com)

Nanobots can be used for diagnostics and investigations, such as:• measurement of blood vessel parameters (temperature, pressure, viscosity);• measurement of chemical parameters (pH, concentration of oxygen, carbon dioxide, presence of antigens, polynucleotides, hormones, neuromediators);• atomic-force scanning of cell surface;• optical and acoustic microscopy;• magnetic-resonance tomography;• electromonitoring of neuron activity, myocytes etc;• chemical monitoring of synapses.


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Nanomaterials -

objects or their compositions possessing new qualities owing to reduction of the object sizes in one, two or three dimensions to nanometer scale. The overwhelming majority of the new physical phenomena in such materials are caused by discretization of properties in quantum- dimensional structures.


Robert Curl (1933)

Richard Smalley (1943 -

2005)

Harold Kroto (1939)

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Fullerens

«bucky

ball»

‐

a class of the carbon

molecules containing more then 20 atoms.

They

have been invented by Richard Smalley, Harold

Kroto

and Robert Curl in

1985. In 1996

these

researchers have been awarded the Nobel Prize in

chemistry.

Carbon nanotubes – lengthy cylindrical structures in diameter from one to several tens of nanometers and length to several centimeters, consist from one or several curtailed into a tube hexagonal graphite planes and ending with usually hemispherical head.


18

The first structures similar to quantum dots (QD) were metal nanoparticles produced in ancient Egypt for a staining of various glasses!

QD with the structure of core and core/shellQD properties:

1. stability;

2. narrow emission spectrum;

3. QD of the given type can be excited by one wavelength;

4. high emission intensity;

5. stability to photochemical and electrochemical destruction.


19

Change of transmission band of colloid solution of CdSe/ZnSe QD is subject to quantum dot diameter

Diameter decreasing


Scanning electron microscopy image

It is shown surgeons' hands in an operating room with a "beam of light" traveling along fiber optics

for photodynamic therapy.

21

Photodynamic therapy (PDT) was developed as a feasible medical technology in the 1980s in several institutions throughout the world. It is a three level treatment for cancer involving three key components: a photosensitizer, light, and tissue oxygen. A photosensitizer

is a chemical compound that can be excited by light of a specific wavelength. This excitation uses visible or near-infrared light.

The German physician Friedrich Meyer–Betz performed the first study with what was called photoradiation therapy (PRT) with porphyrins in humans in 1913.


23

Chromatogram of blood serum of consumptive (solid line) and healthy patients (dotted line)


molecular

mass, kD

Opt

ical

den

sety

, 254

nm

Identification of TB middle mass markers

O.M. Galaichenko, M.M. Rozhitskii. Electrochemiluminescence nanoanalytical device for diagnostics of infectious diseases// Ukrainian-German Symposium on Nanobiotechnology.-Kyiv.–2006.–P. 63.O.M. Galaichenko, M.M. Rozhitskii. Investigation of the Interaction of Amino Acids with Nanoparticles in Electrochemiluminescence Sensor // 3rd ECGEMS Meeting Electrochemistry in “Nanosystems and Molecules at Work”. Czech Republic.–2007.– P. 40.

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A1

A2

A3 A1

A2

A3

A2

A3

A1

A2 A3

A3

A2

A3

A1


1 –working electrode with plotted QD;

2 –

auxiliary electrode; А1

, А2

, А3

–

tuberculosis markers

1 –

QD;2 –

glass substrate;3 –

ІТО

coating

,QDeQD −− →+,AeA +− →−

,AQDAQD * +→+ +−

.hQDQD* ν+→

Electrochemiluminescence – luminescence due to non-optical excitation in charged particles recombination as a consequence of electrochemical and chemical reactions in liquid media

1

32

hν

hν

The Langmuir-Blodgett through (Microtestmachines, Co, Belarus)

1. Substance plotting on a substrate.2. Monolayer compression by the floating

barrier.3. Shift of monolayer to the substrate by its

vertical lifting.


26

Atomic Force Microscope NT-206(Microtestmachines, Co, Belarus)


28

ELAN-3d

Electrochemiluminescent AnalyzerDeveloped by researchers of “Laboratory of Analytical Optochemotronics”


29

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.80

10

20

30

40

50

60

0

100

200

300

400

500

600

Cur

rent

, μA

Potential, V (vs. Ag/AgCl)

Pho

tocu

rren

t, pA

Photocurrent of QD’s which are plotted by LB technology on working electrode of sensor instrument with an addition of 1

мМ

of tuberculosis process marker

Photocurrent of 1

мМ

tuberculosis process marker solution with addition of developed by us new coreactant

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6 1,8

0

100

200

300

400

500

600

700

Phot

ocur

rent

, pA

Potential, V (vs./ Ag/AgCl)


ECL sensor prototype for definition of TB-markers

30

Analytical nanobiophotonic technology

Analytical electrochemiluminescent method

Analytical principle

Sample splitting

Sample preparation

Analytical signal

registration

Analytical data

processing

Subject of investigation

(bioliquid)

Quantity information

about content of

TB markers


31

Data processing

Measured component (analyte) Indirect ECL measurement

Selection of reagent

Direct ECL measurement

Selection of measured instrument

Selection of quantification

method

Determination of analytical

processmechanism

Development of sensor instrument

for measurement of analytical signal

Creation of model system

Resulting information

Measurement of analytical signal


Electrode material glass substrate with ITO

conducting surface

Size, mm 25×15×2,4

Medium for electrolysis dimethylformamide, water

Quantum dots film forming

method of Langmuir- Blodgett

Film’s number 1

Stabilization coating

for water-insoluble quantum dots trioctylphosphine oxide

for water soluble quantum dots thioglycolic acid

LOD for marker’s determination, mol/l 10-9

Duration of probe preparation, min 15

Duration of ECL analysis, min 5 -

10

Selectivity High, depending upon ECL analysis method

Price

Methods to increase selectivity of developed sensor instrument

technologicaltechnological

physicalphysical

electrochemicalelectrochemical

chemical/mechanicalchemical/mechanical


33

Selection of sensor instrument with

needed type and diameter QD

Selection of electrolysis medium

Selection of secondary and

reference electrodes

Addition of solventto electrochemical cell

Attaching to electrode holder of developed sensor instrument

Fixation of electrochemical cell in

ELAN-3dPlacing of lightproof

boxSetting of investigation

parameters

Execution of control measurements without

biological probe

Registration of control ECL signal

Addition into electrochemical cell of

biological probe

Execution of measurements with

biological probe

Registration of analytical signalData processingReport formation

Procedure of electrochemiluminescent investigation of biological

probe with developed nanobiophotonic

sensor instrument


34

1. Science and Technology Center in Ukraine (STCU)1. Science and Technology Center in Ukraine (STCU)STCU Project 4495 “New biophotonic method and nanostructure sensor

instrument for diagnostics of tuberculosis active form”

2. Joint participants from Kharkiv Medical Academy of Postgradua2. Joint participants from Kharkiv Medical Academy of Postgraduate te EducationEducation

3. Colleagues from 3. Colleagues from ““Laboratory of Analytical Laboratory of Analytical OptochemotronicsOptochemotronics””


35

Rozhitskii Rozhitskii MykolaMykola M.,M., General Manager of STCU Projects 4180, 4495, Academician of Academy of Applied Radio Electronics, Professor, Dr. Sci. Phys.-Math., Scientific Chief of Lab. of Analytical Optochemotronics, Professor of Biomedical Electronics Department,

phone +380 57 7020369, fax. +380 57 7021013,e-mail: [email protected]

SnizhkoSnizhko DmytroDmytro V., V., Ph. D. Tech. Sci., Senior Researcher of Lab. of Analytical Optochemotronics, Biomedical Electronics Department, phone +380 57 7020369, fax. +380 57 7021013, [email protected]

Galaichenko Galaichenko AlenaAlena M., M., Scientific researcher of Lab. of Analytical Optochemotronics, Biomedical Electronics Department, phone +380 57 7020369, fax. +380 57 7021013, [email protected]

Our address: Our address: Kharkiv National University of Radio Electronics,Laboratory of Analytical Optochemotronics,Biomedical Electronics Department,61166, Kharkiv, Lenin Av.14, Ukraine.

Our webOur web--site: site: http://electrohim.kture.kharkov.ua/Kharkiv

new biophotonic method and nanostructure sensor instrument ... · robert a. freitas jr.,...

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