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Nanotechnology and diagnosis

Nanotechnology and diagnosis. A.A. 2011-2012. Enhanced PCR. nAu reduces the formation of erroneous copies of DNA in error-prone PCR. M: markers, nAu: 1: 0.6 nm 2: 0.0 nm 3: 0.2 nm 4: 0.4 nm 5. 0.8 nm 6: 1.0 nm. Enhanced staining of cell.

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Nanotechnology and diagnosis

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  1. Nanotechnology and diagnosis A.A. 2011-2012

  2. Enhanced PCR nAu reduces the formation of erroneous copies of DNA in error-prone PCR. M: markers, nAu: 1: 0.6 nm 2: 0.0 nm 3: 0.2 nm 4: 0.4 nm 5. 0.8 nm 6: 1.0 nm

  3. Enhanced staining of cell. Thanks to their colloidal nature, the nanomaterials interacts with the biological systems. Nanoparticles enters the cells and differently accumulate at subcellular level. It is possible to target them to different cell compartments/organelle by selecting their physical-chemistry. In the figure, differently sized QDs stain extracellular space and subcellular structures.

  4. Enhanced “in vivo” imaging: QDs • Functionalized QDs imaging of a rat with muscle sarcoma. • B)The same as A: in this case the QDs did not have been functionalized. • (Cai & Chen, Small, 2007, 3: 1840.)

  5. A B Enhanced “in vivo” imaging: USPIO MRI of brain lesion without (A) and with (B) contrasting with USPIO.

  6. Higher glucose Lower glucose Biochemical monitoring • Nanosensors are implanted into the skin. • They emit fluorescence when irradiated by light. • The fluorescence fades at higher glucose concentrations, because of the splitting of fluorophore by competitive binding. • The same procedure can be applied to measure ions, pH, osmolarity, etc. • Cash & Clark Trends Mol Med. 2010

  7. The Lab-on-a-chip Tseneva & Zhebrun, Pasteur Institute, St. Petersburg, ICPC Nanonet 3rd Annual Workshop, St Petersburg, 24-25 May 2011

  8. How does it work? • Analytes: blood, urine, saliva, gases in tissues, exhaled air. • Integrated processes: sorption, chromatographic separation, electrophoresis, thermocycling, (PCR), light scattering, fluorescence. • Improved methods, implemented with nanotechnologies, • reduces sample volume and handling. • The lab-on-a-chip should save room, time and money. • Advantages: faster, cheaper and more accurate for multiple • analyses.

  9. Main nanostructured components.

  10. Nanoporous Si-based membrane • membrane support structure (A) with 5 µm suspended (free standing) membrane elements (arrow). • Cross section of individual element (arrow) showing the porous nature of the membrane • E,F) Pore shrinking protocol: larger pores are oxidized to be reduced by 50% • G) The pores at their final limit (5 nm) Johannessen et al. 2010. J Diabetes Sci Technol. 4(4):882-92.

  11. Nanoporous Anodic Aluminium Oxide (AAO) membrane The figure shows a nanporous membrane of anodic aluminium oxide (AAO). Lateral (b) and top (c) view. The mean diameter of nanopores is 200 nm. The nanoporous membranes are implemented in devices like those shown in the following slides. Ai et al. 2010 J Colloid Interface Sci. 350(2):465-70. doi: 10.1016/j.jcis.2010.07.024

  12. Nanoporous membranes Nanoporous membrane Sensor (optical fiber) Substrate Signal Complex bio-solution Dialyzed target proteins Modified from: Chang et al. 2008. Nanotechnology. 19(36):365301. doi:10.1088/0957-4484/19/36/365301

  13. Green arrow: constriction of a nanochannel (diameter = 130 nm) Nanochannels The DNA strand enters into a nanochannel (1-3); its compression (4-6) and relaxation (7-9) follow. Reccius et al., 2008. Biophys J 95: 273–286

  14. Separation of a single molecule of DNA into a silica-based nanochannel. + - The sample containing DNA is inserted in a chamber. The electrical field is applied. and assume an elongated equilibrium conformation in the nanochannel The DNA molecules are drawn in the loading zone, enter a nanochannel Modified from: Liu et al., 2011 Electrophoresis, 32: 23: 3308-3318, DOI: 10.1002/elps.201100159.

  15. Nanomixers • The fully assembled device • The 3D structure of the mixer • The serpentine microfluidic channel (50>300 nm) • The structures at the outlet of the mixer • The structures in the middle of the mixer • Cross-sectional view of a structure similar to the ones used in the mixers. Jeon et al. 2005. Nano Letters 5(7): 1351-1356

  16. The mixing process b The the serpentine with (a) and without (b) flow at 13.3 mm/sec. Jeon et al. 2005. Nano Letters 5(7): 1351-1356

  17. Si-based, inner diameter: 100 nm • C-based with nAu coating • C-based, crushproof (a) and injecting a cell (b) Nanopipette • Joe et al. 2006. J. Am Chem Soc 128(51): 16462-16463 . • Bau, Schrlau, Falls and Zinber, University of Pennsylvania School of Medicine. • Bhattacharyya et al. 2009 Adv. Mat. 21(40): 4039-4044 doi: 10.1002/adma.200900673

  18. Nanosensor A sensor based on nanotechnology (Si and metal oxide, STM 90 nm; 90 nm refers to the transistor gate length). Johannessen et al. 2010. J Diabetes Sci Technol. 4(4):882-92.

  19. What could the lab-in-a-chip do? • Cell separation • Rapid cancer detection • DNA analyses, PCR • Microtitration • Blood analyses • Microbiology platforms: growth, identification and classification, antibiotic sensitivity tests • Capillary fluorimetric detection

  20. 1. Loading the blood sample 2. WBC isolation, RBC elution 4. Washing the unbound dye, reading fluo 3. Labeling the WBC with fluo Lymphocyte (WBC) isolation Modified from: Zimina & Lucinin, 2011. J. Anal. Chem. 66(12): 1136–1158.

  21. Rapid cancer detection The hmw DNA (high molecular weigth), a marker for leukemia, is captured from 20 l of untreated blood by the electrophoretic circuit of a chip, activated with a fluorescent green dye. Wash it. On the right (A) the result for an healthy subject, on the left (C) that of a patient with leukemia. (Heller et al., 2011. doi: 10.117/2.1201007.003153)

  22. 1 2 4 DNA analyses, PCR • Heater • SiC thermosensor • Radiator, with the thermosensor shown in red • 4. Separation channel and reservoir for thermocycling. • 5. Pattern of t°C cycles 3

  23. The PCR on-a-chip The nanoporous membrane Modified from: Kim et al. 2010 Analyst. 135(9):2408-14

  24. Continuous glucose measure on-a-chip. Si membrane Johannessen et al. 2010. J Diabetes Sci Technol. 4(4):882-92.

  25. Testing antibiotic sensitivity The picture…. and the scheme Tsou et al. 2010. Biosens Bioelectron. 26(1):289-94.

  26. The results Partial resistance Resistance Sensitivity 0 60 120 180 min Tsou et al. 2010. Biosens Bioelectron. 26(1):289-94.

  27. The goal: the microbiology lab on-a-chip • Culture growth platform • Pre-filter • Sample • Image recognition • Laser detector (LIF) • Colorimeter • Microfluidic sorting element • CDD camera (low resol.) • AST microplatform • Pathogen growth • Bank of antibiotics • Lead • Substrate • Heater • Waste basin • Sorting way Zimina, ICPC NANONET workshop, 24-25 May 2011, St. Petersburg

  28. Another goal: the metabolic lab-on-a-chip Chang, 2004. Proc IEEE, 92(1): 154-173.

  29. Electrophoresis White cell count Emocromo White cell histology Heater Proteins pH Sample well Metabolites Coagulation Reaction centre Another goal: the hematology and clinical pathology lab on-a-chip Kutuzov, St. Petersburg State Electrotechnical University ICPC Nanonet 3rd Annual Workshop, St Petersburg, 24-25 May 2011

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