Nanodevices for the
detection of disease
Molecular diagnostics with nanotechnology
Maurits de Planque
Cafe Scientifique - ...
Diagnostics - diagnosis
Clinical diagnosis
A diagnosis made on the basis of medical signs and patient-reported
symptoms, r...
Diagnostics - molecular diagnostics
Diagnostics = the practice or techniques of diagnosis
Molecular diagnostics:
•  concer...
Diagnostics - pregnancy test strip
Diagnostics - pregnancy test strip
The hormone human chorionic gonadotropin (hCG) is produced during
pregnancy. The test s...
Diagnostics - pregnancy test strip
The test line colours red if hCG-antibody-nanoparticle complexes bind to
a second antib...
Diagnostics - pregnancy test strip
The pregnancy test strip is one of the
best known out-of-laboratory ('point
of care') m...
Diagnostics - preventive medicine
The pregnancy test illustrates the advantages of molecular diagnostics:
•  non-invasive
...
Diagnostics - biomarkers
For molecular diagnostics, we need to have knowledge of biomolecules
that are characteristic for ...
Diagnostics - biomarkers
For complex diseases such as cancer, it is a very active research area to
discover molecular biom...
Diagnostics - biomarkers
Most molecular biomarkers for complex diseases are larger biomolecules:
proteins incl. enzymes an...
Diagnostics - ELISA
The pregnancy test strip works as an immuno-assay, but because it is a
lateral flow assay, it is not t...
Diagnostics - ELISA
An ELISA is carried out by pipetting the various reagents in plastic micro-
wells. This multi-step exp...
Diagnostics - scale of testing
Approximately 1,000,000,000 laboratory tests are performed in the
UK every year = 16 tests ...
up to 400 immuno-assay tests/hour
= 9,600 samples/day
ARCHITECT i4000SR Immunoassay Analyzer
122 x 323 x 125 cm
981 kg
> £...
Cancer
AFP
CA 125
CA 15-3
CA 19-9 XR
CEA
CYFRA 21-1
free PSA
HE-4
proGRP
SCC
total PSA
Neuron-Specific Enolase (NSE)
PIVKA...
Congenitals
CMV IgG
CMV IgG Avidity
CMV IgM
Rubella IgG
Rubella IgM
Toxo IgG
Toxo IgG Avidity
Toxo IgM
Hepatitis
anti-HAV ...
Metabolic
active-B12 (HoloTC)
B12
C-Peptide
Cortisol
Ferritin
Folate
Hemoglobin A1c (whole blood)
Homocysteine
Insulin
int...
Diagnostics - clinical laboratory biomarker tests
Transplant
Cyclosporine
Sirolimus
Tacrolimus
Proteins
anti-CCP
Renal
NGA...
Diagnostics - clinical laboratory testing
This type of equipment is not only expensive in
itself, it is also costly to run...
Electronic engineering is best
known for the fabrication of
integrated circuits such as the
microprocessors that drive
com...
10 transistors
Microprocessors become more powerful year-by-year because the
transistors inside them can be made smaller a...
Micro/nanotechnology - miniaturization
The techniques of microfluidics and laboratory-on-a-chip have
emerged from electron...
Micro/nanotechnology - miniaturized immuno-assay
layout of microfluidic chip with
1,024 reaction chambers500 µm
microscopy...
Micro/nanotechnology - miniaturized immuno-assay
LabChip,2014,14,2642-2650
This miniaturization enables a huge cost saving...
Micro/nanotechnology - point-of-care immuno-assay
Microfluidic tests are also being developed for point-of-care settings,
...
Micro/nanotechnology - point-of-care immuno-assay
0.5 mm 1 mm
The simple signal read-out system costs £49. The reagents co...
Micro/nanotechnology - assays in paper
Microfluidic immuno-assays are also
being implemented in paper, using
two or three-...
Micro/nanotechnology - assays in paper
Microfluidic pathways can be defined
by patterning the paper with photo-
resist (li...
Micro/nanotechnology - mobile phones as detector
Immuno-assays usually generate a colour for positive samples. For
biomark...
Micro/nanotechnology - mobile phones as detector
Biosens.Bioelectron.,2015,64,63-68
(a) 3D printed smartphone accessory co...
Nanotechnology - electrical immuno-assays
The transistors in microprocessors are field effect transistors. These can
be ad...
Nanotechnology - electrical immuno-assays
A change in charge on the surface of the wire can only cause a significant
chang...
Nanotechnology - electrical immuno-assays
Nanowire or nanoribbon field effect transistors are fabricated with the
same tec...
Nanotechnology - nanopore resistive pulse sensing
Another nanotechnology that is being explored for biomarker analysis
is ...
Nanotechnology - nanopore resistive pulse sensing
At Southampton University we are
using the biological nanopore α-
hemoly...
Nanodevices for diagnostics - summary
Summary
•  the early onset of disease will result in changes in the biomolecule
comp...
Nanodevices for diagnostics - summary
Can micro- and nanotechnology deliver cheap biomarker immuno-
assays for routine tes...
Nanodevices for diagnostics - summary
...... so more research and development is required ......
Thank you !
of 39

Nanodevices for the detection of disease by Maurits de Planque

Molecular diagnostics with nanotechnology Presented to Isle of Wight Cafe Scientifique by Maurits de Planque on 9 Feb 2015
Published on: Mar 3, 2016
Published in: Health & Medicine      
Source: www.slideshare.net


Transcripts - Nanodevices for the detection of disease by Maurits de Planque

  • 1. Nanodevices for the detection of disease Molecular diagnostics with nanotechnology Maurits de Planque Cafe Scientifique - Shanklin, Isle of Wight - 9 Feb 15
  • 2. Diagnostics - diagnosis Clinical diagnosis A diagnosis made on the basis of medical signs and patient-reported symptoms, rather than diagnostic tests. Laboratory diagnosis A diagnosis based significantly on laboratory reports or test results, rather than the physical examination of the patient. Radiology diagnosis A diagnosis based primarily on the results from medical imaging studies. Diagnosis: 'The identification of the nature of an illness or other problem by examination of the symptoms'
  • 3. Diagnostics - molecular diagnostics Diagnostics = the practice or techniques of diagnosis Molecular diagnostics: •  concerns diagnostic tests that detect (bio)molecules in blood, urine, etc. •  normally referred to as 'laboratory tests' •  but do not necessarily have to be performed in a laboratory
  • 4. Diagnostics - pregnancy test strip
  • 5. Diagnostics - pregnancy test strip The hormone human chorionic gonadotropin (hCG) is produced during pregnancy. The test strip contains antibodies against hCG. These are labeled with a gold nanoparticle, which gives them a red colour. control line test line mix area Sensors, 2014, 14, 16148
  • 6. Diagnostics - pregnancy test strip The test line colours red if hCG-antibody-nanoparticle complexes bind to a second antibody against hCG. The control line just binds the first nanoparticle-labeled antibody, hence should always become red.
  • 7. Diagnostics - pregnancy test strip The pregnancy test strip is one of the best known out-of-laboratory ('point of care') molecular diagnostics tests. Its advantages are: •  very cheap (paper-based & visual signal) •  very easy to operate and to interpret the result It is a lateral flow assay. But the disadvantages of these lateral flow assays are: •  insensitivity (need a quite a large amount of the analyte) •  not quantitative (just get a "yes" or "no" read-out)
  • 8. Diagnostics - preventive medicine The pregnancy test illustrates the advantages of molecular diagnostics: •  non-invasive (analysis of small samples of urine or blood) •  pre-symptomatic diagnosis (changes in biochemistry precede physical changes) Molecular diagnostics is therefore crucially important for the development of predictive and preventive medicine: •  earliest possible detection of the onset of disease •  diagnosis of disease before physical symptoms emerge •  start treatment of disease before you actually become ill •  improved prognosis and better quality of life •  much smaller cost to the healthcare system
  • 9. Diagnostics - biomarkers For molecular diagnostics, we need to have knowledge of biomolecules that are characteristic for a specific disease: molecular biomarkers. As a (healthy) example, the protein hCG is a molecular biomarker for pregnancy. Elevated levels of PSA (an enzyme) in blood are a molecular bio- marker for prostate cancer. http://dmarknutrition.com/articles/0043.html
  • 10. Diagnostics - biomarkers For complex diseases such as cancer, it is a very active research area to discover molecular biomarkers that, through their presence or through their elevated concentration, are indicative of the onset of the disease. www.molecularmedicineireland.ie/page/g/s/91
  • 11. Diagnostics - biomarkers Most molecular biomarkers for complex diseases are larger biomolecules: proteins incl. enzymes and antibodies. These can only be detected by using antibodies as selective capture probes. Because antibodies are produced by the immune system, the detection experiments are called immuno-assays. the sample (e.g. blood) contains many different biomolecules the antibody only binds the biomolecule (biomarker) of interest biomarker molecules
  • 12. Diagnostics - ELISA The pregnancy test strip works as an immuno-assay, but because it is a lateral flow assay, it is not that sensitive and it cannot quantitate the concentration of a biomarker molecule. Therefore, the gold standard for protein detection and quantification has another format: the enzyme-linked immunosorbent assay (ELISA). In the last step of an ELISA, an enzyme continuously generates a coloured or fluorescent dye. The light intensity of the sample, measured with a sensitive camera, is proportional to the amount of analyte/biomarker in the sample.
  • 13. Diagnostics - ELISA An ELISA is carried out by pipetting the various reagents in plastic micro- wells. This multi-step experiment requires an experienced lab technician.
  • 14. Diagnostics - scale of testing Approximately 1,000,000,000 laboratory tests are performed in the UK every year = 16 tests per person per year. These tests are all performed because there is a reason for concern (i.e. symptoms have emerged) or to monitor an existing health condition. However, for the routine application of predictive and preventive medicine, these tests will have to be performed at a much larger scale. With existing technologies, this is simply too expensive. Using molecular diagnostics for diseases that might occur (pre-symptomatic diagnosis) cannot be afforded.
  • 15. up to 400 immuno-assay tests/hour = 9,600 samples/day ARCHITECT i4000SR Immunoassay Analyzer 122 x 323 x 125 cm 981 kg > £200,000 Diagnostics - clinical laboratory testing Immuno-assay tests of blood or urine (10-150 µL samples) are carried out in clinical laboratories by large automated pipettor robots.
  • 16. Cancer AFP CA 125 CA 15-3 CA 19-9 XR CEA CYFRA 21-1 free PSA HE-4 proGRP SCC total PSA Neuron-Specific Enolase (NSE) PIVKA Cardiac BNP CK-MB Galectin-3 high sensitive Troponin-I Myoglobin Troponin-I Thyroid anti-Tg anti-TPO free T3 free T4 T-uptake total T3 total T4 TSH Tg Diagnostics - clinical laboratory biomarker tests
  • 17. Congenitals CMV IgG CMV IgG Avidity CMV IgM Rubella IgG Rubella IgM Toxo IgG Toxo IgG Avidity Toxo IgM Hepatitis anti-HAV IgG anti-HAV IgM anti-HBc anti-HBc IgM anti-HBe anti-HBs anti-HCV HBeAg HBsAg qualitative HBsAg quantitative HBsAg HBsAg confirmatory HCV Ag Retrovirus anti-HTLV-I/HTLV-II HIV Ag/Ab combo Other Chagas EBV EBNA-1-IgG EBV VCA IgG EBV VCA IgM Syphilis TP Infectious Disease Diagnostics - clinical laboratory biomarker tests
  • 18. Metabolic active-B12 (HoloTC) B12 C-Peptide Cortisol Ferritin Folate Hemoglobin A1c (whole blood) Homocysteine Insulin intact PTH Pepsinogen I Pepsinogen II Vitamin D Therapeutic Drug Monitoring Carbamazepine Digoxin Gentamicin Phenobarbital Phenytoin Theophylline Valproic Acid Vancomycin Methotrexate Diagnostics - clinical laboratory biomarker tests
  • 19. Diagnostics - clinical laboratory biomarker tests Transplant Cyclosporine Sirolimus Tacrolimus Proteins anti-CCP Renal NGAL Fertility/Pregnancy DHEA-S Estradiol FSH hCG (total β-hCG) LH Progesterone Prolactin SHBG Testosterone
  • 20. Diagnostics - clinical laboratory testing This type of equipment is not only expensive in itself, it is also costly to run because it needs a laboratory environment with qualified personnel and because it consumes large amounts of expensive reagents (antibodies etc). The US Medicare 'Clinical Laboratory Fee Schedule' specifies ~£15/test. How can these costs be substantially reduced? •  use smaller sample volumes = consume less reagents •  avoid the necessity of robotic pipettors = simple or no equipment •  avoid expensive optical sensors = use alternative signal read-outs •  develop 'point of care' assay formats (such as the pregnancy strip) that retain the high selectivity and sensitivity of the ELISA test
  • 21. Electronic engineering is best known for the fabrication of integrated circuits such as the microprocessors that drive computers, playstations, tablets, smart phones, etc. Raspberry Pi Micro/nanotechnology - miniaturization iPad 2 microprocessor
  • 22. 10 transistors Microprocessors become more powerful year-by-year because the transistors inside them can be made smaller and smaller. cross-section of Apple A5 microprocessor 1 mm = 1000 µm 1 µm = 1000 nm 1 nm ≈ 10 atoms 60 nm Micro/nanotechnology - miniaturization The semiconductor industry has been pioneering microscale and nano- scale fabrication for decades; the transistors in modern microprocessors are only about 60 nanometer in size. www.chipworks.com
  • 23. Micro/nanotechnology - miniaturization The techniques of microfluidics and laboratory-on-a-chip have emerged from electronic engineering microfabrication methods. Microscale channels are defined by lithography processes and then replicated into a transparent soft plastic, usually the polymer PDMS. These micrometer channels hold nanoliter aqueous solutions; lab-on- a-chip applications of microfluidics aim to perform conventional lab experiments at a much smaller scale with disposable polymer chips. HendAlkhammash
  • 24. Micro/nanotechnology - miniaturized immuno-assay layout of microfluidic chip with 1,024 reaction chambers500 µm microscopy image of two reaction chambers Microfluidic lab-on-a-chip designs are being developed for miniature biomarker immuno-assays. This example from 2014 can measure four protein biomarkers in 1,024 blood samples = 4,096 assays on one microfluidic chip of 2 x 7 cm.
  • 25. Micro/nanotechnology - miniaturized immuno-assay LabChip,2014,14,2642-2650 This miniaturization enables a huge cost saving on assay reagents: However, this microfluidic platform requires that the samples are loaded using complex equipment and the signal read-out needs to be performed with an expensive optical detector array. It still requires a clinical laboratory environment.
  • 26. Micro/nanotechnology - point-of-care immuno-assay Microfluidic tests are also being developed for point-of-care settings, where complex equipment and complicated operation procedures should be avoided. In this example, which has been tested in Rwanda for HIV and syphilis molecular diagnostics, the reagents and the blood sample (1 µL) are sequentially loaded in a tube and then flown through the microfluidic device. This results in colour development for positive samples ("yes/no" test).
  • 27. Micro/nanotechnology - point-of-care immuno-assay 0.5 mm 1 mm The simple signal read-out system costs £49. The reagents cost £0.15 per HIV+syphilis test. A disposable microfluidic cassette (for 8 tests) is £0.10. NatureMedicine, 2011,17,1015
  • 28. Micro/nanotechnology - assays in paper Microfluidic immuno-assays are also being implemented in paper, using two or three-dimensional paper networks. The fluid flow is capillary. Anal.Chem.,2014,86,6447-6453 malaria test
  • 29. Micro/nanotechnology - assays in paper Microfluidic pathways can be defined by patterning the paper with photo- resist (lithography) or wax (printing). PNAS,208,105,19606-19611 S
  • 30. Micro/nanotechnology - mobile phones as detector Immuno-assays usually generate a colour for positive samples. For biomarkers that are present at low concentration in the sample, this signal can be too weak to detect by eye, especially in small microfluidic chips. However, these chips can be attached to mobile phone cameras. LabChip,2014,14,3159-3164
  • 31. Micro/nanotechnology - mobile phones as detector Biosens.Bioelectron.,2015,64,63-68 (a) 3D printed smartphone accessory comprising a plano-convex lens holder and the lateral flow assay cartridge adaptor. (b) 3D printed cartridge, housing the strip with control (C) and test (T) lines. (c) The integrated cortisol assay smartphone- based device with running application for chemiluminescence signal acquisition. Cortisol (~stress/depression) in saliva can be quantified between 0.3–60 ng/mL.
  • 32. Nanotechnology - electrical immuno-assays The transistors in microprocessors are field effect transistors. These can be adapted to work as biosensors for biomarker detection. •  a small electrical current flows through a semiconductor wire that is positioned between two electrodes •  the wire has antibodies against a particular biomarker attached to it •  when the biomarker in the sample becomes bound to the antibodies, the amount of charge at the surface of the wire changes •  this causes a change in the electrical current flowing through the wire
  • 33. Nanotechnology - electrical immuno-assays A change in charge on the surface of the wire can only cause a significant change in the current flowing through the wire if the diameter of the wire is very small. The wire should have a diameter of only ~50 nanometer, or if a ribbon is used, the ribbon should have a thickness of only ~20-50 nm. At Southampton University, we are developing silicon oxide nanoribbons and zinc oxide nanowires as nano- scale field effect transistors for the analysis of biomarker proteins. Nano Letters, 2012, 12, 1868-1872
  • 34. Nanotechnology - electrical immuno-assays Nanowire or nanoribbon field effect transistors are fabricated with the same technology as used for microprocessors, hence the cost per nanowire chip will be minimal when the chips are mass-produced. Read-out of the biomarker detection signal would only require an electrical detector such as the glucose meter, avoiding the use of costly optical systems.
  • 35. Nanotechnology - nanopore resistive pulse sensing Another nanotechnology that is being explored for biomarker analysis is nanopore resistive pulse sensing. When a biomarker molecule that is bound to a selected receptor molecule passes through the pore, the pore is blocked for a short time, which shows as a downward current spike: i blocked. Ions are flowing through a nanopore, which gives a nano-Ampere electrical current: i open. Chem. Rev., 2012, 112, 6431-6451
  • 36. Nanotechnology - nanopore resistive pulse sensing At Southampton University we are using the biological nanopore α- hemolysin (1.4 nm pore width) for resistive pulse sensing experiments. A larger number of downward current spikes indicates a higher concentration of the biomarker in the sample. This assay may be particularly suitable for the detection of microRNA molecules. These are emerging as a new family of biomarkers that could be specific for different cancer types. miRNA-DNA duplex αHL nanopore artificial cell membrane
  • 37. Nanodevices for diagnostics - summary Summary •  the early onset of disease will result in changes in the biomolecule composition of blood and urine etcetera, before symptoms emerge •  laboratory tests can detect and quantify these biomarker molecules •  biomarker detection can result in an early diagnosis and therefore an improved prognosis; prevention of disease progression •  preventive or predictive medicine is a sustainable healthcare model •  biomarker tests for complex diseases tend to be immuno-assays •  presently, most assays can only be performed in clinical laboratories •  there is no (financial) scope for performing such tests on a routine basis, which would require a very large number of tests per person However:
  • 38. Nanodevices for diagnostics - summary Can micro- and nanotechnology deliver cheap biomarker immuno- assays for routine testing for many different complex diseases? •  this is a very active cross-disciplinary research area •  there are many different technologies being explored •  some promising results have been achieved •  however, as yet, none of these systems combines all the advantages offered by the expensive automated robotic immuno-assay platforms How can the cost of biomarker testing be substantially reduced? •  use smaller sample volumes = consume less reagents •  avoid the necessity of robotic pipettors = simple or no equipment •  avoid expensive optical sensors = use alternative signal read-outs •  develop 'point of care' assay formats (such as the pregnancy strip) that retain the high selectivity and sensitivity of the ELISA test
  • 39. Nanodevices for diagnostics - summary ...... so more research and development is required ...... Thank you !

Related Documents