Abel González Oliva
Laboratório Diagnóstico Biomolecular
ITQB-UNL / IBET
JORTEC – WORKSHOP : BIOSSENSORES
FCT-UNL 21/02/2013
• Imunoensaios e imunosensores
• Testes rápidos
• Sensores enzimáticos
• Sensores baseados em células
• Sensores de DNA (DNA-chips)
• Instrumentos para a saúde humana
• Instrumentos para aplicação em
biotecnologia
The affinity and specificity of an antigen - antibody interaction to form a
stable complex, are the basis properties for an immunoassay
antibody
antigens
support
label
Immunoassay: A laboratory technique that makes use of
the binding between an antigen and its homologous
antibody in order to identify and quantify the specific
antigen or antibody in a sample.
Imunoensaio
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
An assay that quantifies antigen or antibody by immunochemical means.
An antigen can be a relatively simple substance such as a drug, or a complex one
such as a protein or a virus.
• Immunofluorescence
• Immunonephelometry
• Radioimmunoassay
Immunofluorescence
human skin prepared for direct immunofluorescence
Imunoensaio
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Antigens from the sample are attached to a surface of a plate (e.g. 96 wells). Then, a
specific antibody is applied over the surface so it can bind to the antigen. This antibody is
linked to an enzyme, and, in the final step, a substance (enzyme’s substrate) is added.
The subsequent reaction produces a detectable signal, most commonly a color change,
quantified by the ELISA reader.
ELISA (Enzyme-linked immunosorbent assay)
ELISA, Enzyme Linked Immuno Assay is a biochemical technique used
mainly to detect the presence of an antibody or an antigen in a sample.
The ELISA has been used as a diagnostic tool in clinical medicine, plant
pathology, environmental monitoring, as well as a quality control check
in various industries.
ELISA reader. 96 wells polystyrene microtiter plate
Plate well
E E E E
S S S S P
P P P
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Immunosensors transduce antigen-antibody interactions directly into physical
signals. The design and preparation of an optimum interface between the
biocomponents and the detector material is the key part of sensor development.
Immunosensors can be applied in diagnostic of diseases in clinical health of
humans and animals; in the detection of drugs and environmental pollutants
and in the food quality control, allowing applications that need a rapid
detection with high sensitivity and specificity.
Imunosensor
antibody
antigens
support
label blocking agent
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
• Antibodies are proteins
• Synthesized by plasma cells, i.e. mature B-lymphocytes
• Response to immunogen
• Immunoglobulins (G, A, M, D, and E)
• Immunoglobulin G (IgG), also known as -globulin, 150 kd
• Principal antibody in serum.
Antibodies
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Immunosensors
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Immunosensors
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Immunosensors
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
DNA-chip
A DNA microarray is used in molecular biology and consists of an arrayed series of thousands
of microscopic spots of DNA oligonucleotides, each containing picomoles (10−12 moles) of a
specific DNA sequence, known as probes (or reporters).
Example of an approximately 37,500 probe spotted oligo
DNA-chip
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
DNA-chip
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
High density, oligonucleotide-based DNA arrays.
DNA-chip
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Sensores baseados
em células
Sensores baseados em células
Cell-based biosensors (CBBs) have emerged as powerful functional tools for
the rapid detection of hazards and threats associated with food, agriculture,
environment and biosecurity.
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Relative advantages of cell-based sensors
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Relative disadvantages of cell-based sensors
• cells to respond to complex mixtures
• Receptors, channels, and enzymes active
• Alternatives to animal testing
• Better tolerance for solute and pH variations
• Longer life than enzyme biosensors
• Need optimized environment for long periods to be effective
• Need physical and chemical controlled conditions
• Slower reaction / measurement time than biosensors
• Longer Recovery time
Sensores baseados em células
hazard-induced
physiological responses
receptor–ligand interactions, signal transduction,
gene expression, membrane damage, apoptosis
and oncosis of living sensing organisms
toxicity
array-formatted impedance biosensor
(a) The biosensing chip with a microelectrode array and cell-culture
wells. (b) Cells on the electrode, (left) before and (right) after attachment.
Whole cell based electrical impedance sensing approach for a rapid nanotoxicity assay. Evangelia
Hondroulis et al 2010 Nanotechnology 21 315103 doi: 10.1088/0957-4484/21/31/315103
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Sensores baseados em células
Field Effect Transistor (FET)
Peter Fromherz. Solid-State Electronics. Volume 52, Issue 9, September 2008, Pages 1364-1373
Papers Selected from the 37th European Solid-State Device Research Conference - ESSDERC’07
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Sensores baseados em células
Real-time measurements of the shift in resonance frequency and energy dissipation due to
changes in mass and viscoelastic properties give quantitative information about the state
of the cell adhesion process
(D. Johannsmann, Viscoelastic analysis of organic thin films on quartz resonators - 2000)
Quarz Crystal Microbalance (QCM)
J.Elsom, M.I.Lethem, G.D. Rees and A. Christy Hunter. Novel quartz crystal microbalance
based biosensor for detection of oral epithelial cell–microparticle interaction in real-time
Biosensors and Bioelectronics 23(8)1259-1265, 2007 DOI:10.1016/j.bios.2007.11.020
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Sensores baseados em células
Light Addressable Potentiometric Sensor (LAPS)
Myocytes
or neurons
pH-sensitive LAPS that uses LEDs in combination with (semiconducting) silicon and pH-sensitive Ta2O5.
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Sensores baseados em células
cell- and tissue-based sensors
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Sensores enzimáticos
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Sensores enzimáticos
substrate product
Enzyme
Specificity
Selectivity
Change in proton conc.
Release or uptake of gas
Light emission, absortion or
reflectance
Heat emission…
cofactor
Reaction catalized
by the enzyme
Transducer
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
two possibilities to measure the glucose concentration: oxygen consumption or the
hydrogen-peroxide production. As a third option it would be also possible to measure the
temperature increase due to heat released by the two reactions.
The presence of the second enzyme (catalase), although not really essential for the assay,
enhances the first reaction. This happens due to the decomposition of the hydrogen-
peroxide.
Sensores enzimáticos
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Enzymes used in different biosensors based in enzymatic inhibition
Sensores enzimáticos
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Testes rápidos
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Testes rápidos
In vitro diagnostic test strips based on the principles of immunochromatography exist
for a wide array of target analytes. The first tests were made for the detection of
human chorionic gonadotropin (hCG).
Today, there are commercially available tests for monitoring ovulation, detecting
infectious disease organisms, analyzing drugs of abuse, and measuring other analytes
important to human physiology. Products have also been introduced for veterinary
testing, agricultural applications, environmental testing, and product quality evaluation.
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Most immunochromatographic test strips are modeled after existing immunoassay
formats. Thus, test strips for hCG are typically sandwich assays; and drugs of
abuse are analyzed by competitive or inhibition assays. In serum assays,
antibodies are detected as an indicator of various disease states.
Many variations are possible, but they all have in common the formation of a
complex between a detector particle that is free in the sample stream and a capture
reagent that is bound to the membrane at the test line.
Testes rápidos
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Testes rápidos
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
mLabsTM Cardiac Panel
based on immunoassay
technique, is a point-of-care
(POC) diagnostic chip to
diagnose acute myocardial
infarction. It can simultaneously
detect three myocardial markers
including creatine kinase-MB,
myoglobin, and Troponin
General Description:
Visitect Syphilis test, kit of 25 tests.
Technical Specifications:
Technology: Immunochromatographic
Antigen type: TpN17, TpN47
Solid phase: Membrane strip in cassette
Specimen type: Whole blood/plasma/serum
Volume of sample: 50 µl whole blood or 25 µl serum/plasma
Results available for reading: 15 minutes
Shelf life: 24 months at +4° to +30°C.
Temperature requirements for storage: +4° to +30°C –
DO NOT FREEZE.
Testes rápidos
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Instrumentos para
a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
•optical spectroscopy
•mass spectrometry
•electrophoresis and other
separation techniques
•microscopy
•lasers and optics
•X-ray tools and techniques
•nuclear magnetic resonance
spectroscopy
Biomedical Applications
•bioreactors and other forms of cell
culture
•proteomics
•genomic sequencing
•functional genomics
•comparative genomics
•microarrays
•human sequence variation (e.g.
genotyping).
•biosensors (POC)
Ceramic Foams
Instrumentos para a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
epidermal electronics
•Lysine
•Acetylcholine
•Uric acid
•Urea
•Glutamate
•Ethanol
•DNA diagnostics
Disease diagnostic – Clinical analysis - Intensive care monitorization
•Viral diseases
•Bacterial diseases
•Genetic diseases
•Cancer
•Diabetes glucose
•Lactate
•Cholesterol
Clinical Diagnosis
Instrumentos para a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Eestimated 554 million adults in 2007
Instrumentos para a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Oxidase enzymes such as
Gox act by oxidising their
substrates, accepting
electrons in the process
and thereby changing to
an inactivated reduced
state. These enzymes are
normally returned to their
active oxidised state by
transferring these
electrons to molecular
oxygen, resulting in the
production of hydrogen
peroxide (H2O2)
Many inorganic redox
couples (mediators) and
organic dyes have been
successfully utilised as
electron sinks for the
glucose oxidase catalysed
oxidation of glucose.
These redox couples, or
mediators, are able to
shuttle electrons between
the redox centre of the
enzyme and the electrode.
J.D. Newman, A.P.F. Turner / Biosensors and Bioelectronics 20 (2005) 2435–2453
Instrumentos para a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Subcutaneously implantable glucose sensor
Miniature needle-type
amperometric glucose sensors
The Guardian RT system, recently approved by the U.S. Food and Drug Administration for
patients over the age of 18 with Type 1 and Type 2 diabetes, is a doctor-prescribed, patient-
owned continuous glucose monitoring system that displays an updated REAL-Time glucose
value every five minutes and alerts patients when glucose levels become too high or too low,
this information helps patients take action before they experience a problem.
Instrumentos para a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Cocaine
Meth-amphetamines
Ecstasy
Opiates
THC (cannabis)
TNT explosives
Tetryl
DNT
RDX
Nitroglycerin
PETN
The commercial BIOSENS® detects:
BIOSENS® 300
for analysis of narcotics
and explosives
Immunosensors
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
High Sensitivity detection using a zero reflectance
condition on a coated silicon wafer. A) Thin film
schematic and modeling variables. B) Simulated
reflectance of a silicon dioxide layer on Silicon in an
air ambient for 633 nm laser light – the zero
condition exists for a 1419 Å thick film at 70.7°
incidence. C) biodetection methodology – molecular
binding causes a strong reflactance increase
Experimental AIR setup and reference images. A) The
low-complexity AIR imaging reader. B) Reference
image of a 264 spot photolithographically patterned
array. C) Imaging reference chips are fabricated using
photolithography and highly accurate etching to form
arrays of spots slightly thicker than the remaining film.
Images of arrays with spots heights of 700 pm and 10
pm are shown. D) Relative sizes of human serum
albumin (HSA), FITC dye, and water, compared to a 10
pm step height. (i.e., smal fractions of a mollecular
monolayer can be imaged).
Adarza BioSystems, Inc Rapid and label-free biological assay platform for
measuring clinical and point-of-care (POC) samples.
Instrumentos para a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
microfluidics, magnetic particles and gold nanoparticles encoded with
antibodies and DNA (Virigene )
Instrumentos para a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Instrumentos para a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Processor SP
Reader
Nanosphere
VERIGENE
Encapsulated Carbon Nanotubes for Implantable
Biological Sensors to Monitor Blood Glucose Levels
Near-infrared nanoscale sensor that detects
glucose. The sensor could be inserted into
tissue, excited with a laser pointer, and
provide real-time, continuous monitoring of
blood glucose level. The hydrogen peroxide
changes the optical properties of the
nanotubes so that the more hydrogen
peroxide present, the more the tubes will
fluoresce when exposed to near-infrared laser
light.
University of Illinois at Urbana-Champaign and U. Stanford (CA)
Glucose oxidase is sensitive to glucose between 0-10 mmol but, in combination with multi-walled
carbon nanotubes, its detection sensitivity extends to 20 mmol. This range covers the
physiologically relevant values for diabetes.
Instrumentos para a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
New sensors are based on single-walled carbon nanotubes: cylindrical
molecules whose sides are formed from a lattice of carbon atoms This
glass capillary tube, shown here on a fingertip, has been loaded with
glucose-sensitive nanotubes. The capillary tube keeps the nanotubes
confined, but has porous walls so that glucose molecules can get to
them.
The idea is to exploit the nanotubes’ ability to fluoresce, or glow, when illuminated by certain
wavelengths of infrared light—“a region of the spectrum where human tissue and biological fluids
are particularly transparent,” (Michael S. Strano, University of Illinois in Urbana-Champaign )
Instrumentos para a saúde humana
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
World market
for glucose
biosensors and
test strips 11,5
billions in 2012
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Instrumentos para a saúde humana
Diabetes
world prevalence of diabetes among adults
(20–79 years of age) would be 6.4%,
affecting 285 million adults in 2010
and estimated 439 million adults by 2030
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Instrumentos para a saúde humana
Instrumentos para aplicação
em biotecnologia
Fermenters
Production and Quality control of final Products
Monitoring and Process control
•Biosensor for Industrial Pharmaceutical Research Applications
•Protein Chip-based Biosensor
Optical Biosensors for Protein Studies
Membrane Biosensor
Lab-on-a-chip based Biosensor
Special Purpose Biochip-based Biosensors
Instrumentos para aplicação em biotecnologia
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Food Industry Biosensor Applications
Food Safety Monitoring
Rapid Food Diagnostics and Biosensors
Biosensor Technologies that Apply to Food
Single Function-Type Enzyme Sensor
Alcohol Sensor
Glucose Sensor
Lactic Acid Sensor
Peptide Sensor
Multi-Function-Type Enzyme Sensor
Fish & Shellfish's Freshness Sensor
Smell or Taste Ingredient Sensor
Monitoriztion of food and drinks
Food Safety Threats
Botulism
Salmonellosis
Escherichia coli O157:H7
Shigella dysenteriae Type 1
Typhi (typhoid fever)
Vibrio cholerae (cholera)
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Attana AB
Quality control of final Products
Consumption of hormone-treated beef may be causing girls to reach
puberty earlier than they used to and making them more susceptible to
breast cancer, say researchers attending a world conference on breast
cancer.
Pediatricians say the onset of menstruation has steadily decreased in
recent decades. The average age for a first period is now 12½, up from
age 14 in 1900.
Hormones are used by cattle farmers in Canada and the United States
to increase the weight of cattle prior to slaughter. They are currently
the focus of a major trade dispute between North American and the
European Union.
Antibiotics used in chicken: After a European Union ruling on
July 1, only two chemicals - flavomycin and avilamycin - are
used to speed up growth. Until then several antibiotics were
routinely used for more than 20 years on poultry and pigs.
Antibiotics are used in pigs to prevent the spread of disease
in often cramped conditions. Their defenders argue that
chickens grow 5% faster, and the 3-4% saved on feed costs
is vital in keeping businesses viable.
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL
Medical
BioDefense
Food &
Beverage Environment
Bio/Pharma
Research Biosensor
Market
Biosensor market
JORTEC - WORKSHOP EM BIOSSENSORES 21/02/2013 Abel G. Oliva Lab. Diagnostico Biomolecular/ITQB-UNL