3MN170 - Summary
Doorslaer, R.F.J.F. Van
Molecular Biosensing
1/21/21
,Lecture 1 – Introduction molecular biosensing ...................................................................................... 2
Lecture 2 – Molecular recognition .......................................................................................................... 6
Lecture 3 – Nucleic acid diagnostics ..................................................................................................... 14
BZ – week 2a ......................................................................................................................................... 17
Lecture 4 – Immunoassay principles and (particle) labels .................................................................... 19
BZ – week 2b ......................................................................................................................................... 22
Lecture 5 – Diagnostic and analytical metrics, kinetics ........................................................................ 25
BZ – week 3a ......................................................................................................................................... 27
BZ – week 3b ......................................................................................................................................... 30
Lecture 7 - Single molecule detection and plasmonic .......................................................................... 32
BZ – week 4a ......................................................................................................................................... 35
Lecture 8 – Plasmonic biosensors and application ............................................................................... 40
Lecture 9 – Optical detection with magnetic particles ......................................................................... 41
BZ – week 5a ......................................................................................................................................... 43
Lecture 10 – Tethered particles with surfaces ...................................................................................... 46
BZ – week 5b ......................................................................................................................................... 48
Lecture 11 – Biosensing based on Electrical Detection ........................................................................ 51
Lecture 12 – In-vivo molecular biosensing ........................................................................................... 53
BZ – week 6b ......................................................................................................................................... 56
1
,Lecture 1 – Introduction molecular biosensing
Biosensor definition
IUPAC recommendations 1999: “A biosensor is an independently integrated receptor transducer
device, which is capable of providing selective quantitative or semiquantitative analytical
information using a biological recognition element”
Biomarker definition
National Institute of Health 1998: “A biomarker is a characteristic that is objectively measured and
evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic
responses to a therapeutic intervention.”
- A biomarker can be referred to as antigen / analyte / target, depending on the context.
- DNA / RNA biomarkers are used for genetic inherent diseases.
- Proteins are important class of biomarkers.
- Many small molecules and ions are also candidate biomarkers.
The presence of biomarkers is measured mostly in blood plasma.
Blood : hematocrit (PCV= packed cell volume, consists of erythrocytes, leukocytes and platelets) +
plasma (= serum + fibrinogen, liquid consist 7 vol% of proteins, ~ 10^5 different proteins)
Biomolecular separation techniques
Gel electrophoreses
1. Agarose gel electrophoresis
a. DNA and protein separation
b. Large protein concentrations are needed
c. Not very sensitive
d. Agarose → non-uniform pore sizes
2. PAGE (polyacrylamide gel electrophoresis)
a. Polyacrylamide → uniform pore sizes
b. Native PAGE
i. Native conformation (and thus enzymatic functions) of protein is preserved
c. Denaturizing PAGE
i. proteins (SDS, sodium dodecyl sulphate), detergent or DTT for reducing)
3. 2D gel‐electrophoreses
a. Separation on protein charge‐isoelectric focusing (IEF), pI (PIE)‐ isoelectric point is
the pH at which a molecule carries no net electrical charge or is electrically neutral in
the statistical mean. (This is used to separate proteins initially).
b. Separation on molecular weight (size)
Chromatography
Principle: Separation by retention time in column of porous silica particles with hydrophobic coating
in a pressure gradient.
Only low protein concentrations are needed, which make these very sensitive techniques. →
“molecular fingerprint”.
1. High performance liquid chromatography (HPLC)
a. Most used: Reverse Phased HPLC (RP-HPLC) → hydrophobic coating (stationary
phase).
2
, 2. Mass Spectrometry (MS)
a. Combined with different ionization techniques like MALDI or ESI.
b. MALDI‐TOF= Matrix Assisted Laser Desorption Ionization ‐ Time of Flight MS
Discovery of biomarkers
Screening increase in protein expression during diseases
Blotting techniques
- Southern Blotting (DNA)
- Northern Blotting (RNA)
- Western Blotting (Proteins)
o Transfer separated proteins onto membrane (transport through gel by electric field
or capillary forces)
o Add antibody specific to certain protein on membrane
o Detection with labelled secondary antibody (enzymes or chromophores)
High-throughput screening
Large scale screening of hundreds and thousands of biomolecules, screening for increased
expression levels.
1. Recognition molecule attached to substrate (coated slides or membranes):
a. Oligonucleotides (screen DNA, RNA levels),
b. Aptamer (reacts selective to protein or DNA sequence),
c. Antibodies (Ab, often used for proteins)
d. Peptides
2. Followed by a (functional ) detection step : secondary Ab or aptamer with fluorescent tag.
Important biological fluids containing biomarkers
- Blood plasma (most used)
- Urine (biomarker levels low, 'polluted' with other components, not a lot disease products
end up in urine)
- Saliva
- Cerebro Spinal Fluid (CSF)
- Biopsy / tissue / interstitial fluids
The basic or comprehensive metabolic panel (BMP and CMP) in blood analysis
General tool to evaluate metabolism:
Organ function, fluid balance, diabetes, liver and kidney failure → Detection at mM concentrations,
lacks specificity.
3
Doorslaer, R.F.J.F. Van
Molecular Biosensing
1/21/21
,Lecture 1 – Introduction molecular biosensing ...................................................................................... 2
Lecture 2 – Molecular recognition .......................................................................................................... 6
Lecture 3 – Nucleic acid diagnostics ..................................................................................................... 14
BZ – week 2a ......................................................................................................................................... 17
Lecture 4 – Immunoassay principles and (particle) labels .................................................................... 19
BZ – week 2b ......................................................................................................................................... 22
Lecture 5 – Diagnostic and analytical metrics, kinetics ........................................................................ 25
BZ – week 3a ......................................................................................................................................... 27
BZ – week 3b ......................................................................................................................................... 30
Lecture 7 - Single molecule detection and plasmonic .......................................................................... 32
BZ – week 4a ......................................................................................................................................... 35
Lecture 8 – Plasmonic biosensors and application ............................................................................... 40
Lecture 9 – Optical detection with magnetic particles ......................................................................... 41
BZ – week 5a ......................................................................................................................................... 43
Lecture 10 – Tethered particles with surfaces ...................................................................................... 46
BZ – week 5b ......................................................................................................................................... 48
Lecture 11 – Biosensing based on Electrical Detection ........................................................................ 51
Lecture 12 – In-vivo molecular biosensing ........................................................................................... 53
BZ – week 6b ......................................................................................................................................... 56
1
,Lecture 1 – Introduction molecular biosensing
Biosensor definition
IUPAC recommendations 1999: “A biosensor is an independently integrated receptor transducer
device, which is capable of providing selective quantitative or semiquantitative analytical
information using a biological recognition element”
Biomarker definition
National Institute of Health 1998: “A biomarker is a characteristic that is objectively measured and
evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic
responses to a therapeutic intervention.”
- A biomarker can be referred to as antigen / analyte / target, depending on the context.
- DNA / RNA biomarkers are used for genetic inherent diseases.
- Proteins are important class of biomarkers.
- Many small molecules and ions are also candidate biomarkers.
The presence of biomarkers is measured mostly in blood plasma.
Blood : hematocrit (PCV= packed cell volume, consists of erythrocytes, leukocytes and platelets) +
plasma (= serum + fibrinogen, liquid consist 7 vol% of proteins, ~ 10^5 different proteins)
Biomolecular separation techniques
Gel electrophoreses
1. Agarose gel electrophoresis
a. DNA and protein separation
b. Large protein concentrations are needed
c. Not very sensitive
d. Agarose → non-uniform pore sizes
2. PAGE (polyacrylamide gel electrophoresis)
a. Polyacrylamide → uniform pore sizes
b. Native PAGE
i. Native conformation (and thus enzymatic functions) of protein is preserved
c. Denaturizing PAGE
i. proteins (SDS, sodium dodecyl sulphate), detergent or DTT for reducing)
3. 2D gel‐electrophoreses
a. Separation on protein charge‐isoelectric focusing (IEF), pI (PIE)‐ isoelectric point is
the pH at which a molecule carries no net electrical charge or is electrically neutral in
the statistical mean. (This is used to separate proteins initially).
b. Separation on molecular weight (size)
Chromatography
Principle: Separation by retention time in column of porous silica particles with hydrophobic coating
in a pressure gradient.
Only low protein concentrations are needed, which make these very sensitive techniques. →
“molecular fingerprint”.
1. High performance liquid chromatography (HPLC)
a. Most used: Reverse Phased HPLC (RP-HPLC) → hydrophobic coating (stationary
phase).
2
, 2. Mass Spectrometry (MS)
a. Combined with different ionization techniques like MALDI or ESI.
b. MALDI‐TOF= Matrix Assisted Laser Desorption Ionization ‐ Time of Flight MS
Discovery of biomarkers
Screening increase in protein expression during diseases
Blotting techniques
- Southern Blotting (DNA)
- Northern Blotting (RNA)
- Western Blotting (Proteins)
o Transfer separated proteins onto membrane (transport through gel by electric field
or capillary forces)
o Add antibody specific to certain protein on membrane
o Detection with labelled secondary antibody (enzymes or chromophores)
High-throughput screening
Large scale screening of hundreds and thousands of biomolecules, screening for increased
expression levels.
1. Recognition molecule attached to substrate (coated slides or membranes):
a. Oligonucleotides (screen DNA, RNA levels),
b. Aptamer (reacts selective to protein or DNA sequence),
c. Antibodies (Ab, often used for proteins)
d. Peptides
2. Followed by a (functional ) detection step : secondary Ab or aptamer with fluorescent tag.
Important biological fluids containing biomarkers
- Blood plasma (most used)
- Urine (biomarker levels low, 'polluted' with other components, not a lot disease products
end up in urine)
- Saliva
- Cerebro Spinal Fluid (CSF)
- Biopsy / tissue / interstitial fluids
The basic or comprehensive metabolic panel (BMP and CMP) in blood analysis
General tool to evaluate metabolism:
Organ function, fluid balance, diabetes, liver and kidney failure → Detection at mM concentrations,
lacks specificity.
3
