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MAE 6291 Biosensors and Bionanotechnology
Format
lecture, discussion, lots of questions
will aim to have students present
segments of papers in each class (.25)
homework ~1 every 2-3 classes to learn
how to use what we cover (.25)
and help analyze papers
occasional demonstrations – e.g. ELISA,
fluorescence microscopy, pcr
take-home midterm exam (.25)
take-home final exam
or student presentation (.25)
Goals –
1. learn about nanotechnology-based biosensors
molecules (analytes) detected
molecules used to provide specificity
transducing modalities (light, mass, electricity)
assay formats (sandwich, labels, label-free)
processes affecting time to get signal (diffusion,
binding kinetics) and sensitivity
multiplex methods (e.g. hybridization arrays)
massively parallel DNA sequencing methods
clinical significance of assays
More Goals
2. Quantitative understanding of relevant nanoscale
processes and phenomena, including Brownian
motion, reaction kinetics, mechanical properties of
biopolymers like DNA at the single-molecule level
3. Understand how some subcellular biological systems, like
molecular motors, transduce chemical energy
into motion
4. Appreciate overlap between engineering and biology
5. Gain experience reading research papers critically
Contact info:
[email protected], tel 240 447 3268
set up time to meet for office hours
Much better to meet often to go over questions early
References for class 1
Philip Nelson Biological Physics
Ch 1, 1.4-1.5 Dimensional analysis, molecules pp. 18-29
Ch 2, 2.2 Molecular Parts List, pp.45-62.
Molecules (things) to be detected and how they interact
ions
small molecules (MW < 600g/mole=10-21g,
or ~50 atoms – e.g. glucose)
peptides – short string of amino acids
proteins – string(s) of up to ~1000 amino acids
viruses - ~1000+ proteins + NA genome (>104 bases)
oligonucleotides – short string of nucleic acids
= bases A, G, C, T (U) – joined via sugar-PO4
nucleic acid sequence
Ions –
e.g. Na+, K+, Mg++, Cl-, PO4—
typical size?
In solution: typical concentration, 1-100mM
units: 1M = NA/liter = 6x1023/10-3m3
how many is that /cm3 or ml?
how far apart are they?
Why do they move?
How will they be distributed near charged objects?
Typical distances over which fixed charges are shielded
Debye length
=.3nm/I1/2 (I in M)
What does this mean in terms of electrostatic interactions?
Small molecules – e.g. sugars, < 100 atoms, size? (~1nm)
H, O, C = hydrogen, oxygen,
carbon atoms, etc.
Vertices = C atoms (understood)
Lines = covalent bonds
strength ~eV (1.6x10-19J)
What is significance of glucose in biology/medicine?
Diabetes – does it go up or down?
problems if it goes up
problems if it goes down
More on units
Molecular weight = weight of NA (6x1023) molecules
(=1 mole) in grams
H has molecular weight =1g/mole
C “weighs” 12 g/mole
“Small” molecules defined as above have MWs ~ or <500
Aside on energy scales
molecules always jiggling in water
Average energy of molecule, each “mode” of
interaction, e.g. translation, vibration between atoms
= kBT (4x10-21J at room temp = 1/40th ev)
Do all molecules have average energy in solution?
What is probability that a molecule has energy E?
Boltzman distribution: p ~ exp(-E/kBT)
What is relative probability that a sugar molecule
hit by a particularly energetic water molecule
at room temperature will get enough energy
to break a covalent bond?
p ~ exp(-40kBT/kBT) = 10-18
So are covalent bonds usually stable at room temp.?
Another class of small
molecules
All NH2-CHX-COOH
side groups X differ
some have + or –
charge
others partial charge
others hydrophobic
“greasy”
-> weak interactions
(~kBT) w/ other
molecules
Protein = linear polymer of amino acids (aa)
chains from a few (“peptide”) to ~1000 aa long
MWs ~100,000 g/mole (aka “kiloDalton”, kDa)
Protein polymers “fold up” into fairly compact units
~10nm, based on weak interactions between
amino acids
Some proteins fairly rigid = “fixed” structure
often known from crystallography
Others don’t crystallize, probably “floppy”
(or have parts that are floppy) in solution
Some have a few, alternative “rigid” shapes (important!)
Surface distribution of charged, polar
(partially charged), hydrophobic, etc
groups -> specific interactions with other
molecules
Note how different from usual physics – gazillions
of identical electrons interacting uniformly
Glucose oxidase ~ 600 aa protein enzyme that binds and
oxidizes glucose. Ribbon model of its aa backbone, portions of which form helices. Note size, complexity relative
to glucose, a simple sugar typical of small molecule targets
~ 3 nm
Model of a particular protein showing charged
surface regions (red -, blue +), and some drug molecules
in binding pockets. Note complexity of surface
allowing complex interaction with other molecules
http://www.pnas.org/content/104/1/42/F6.ex
pansion.html
Proteins can interact forming larger polymers
(of polymers) –> structural elements like
fibers of collagen or microtubules (~25nm in
diameter, microns long)
Proteins also can act as enzymes, “catalyzing”
chemical reactions that break and reform
covalent bonds
http://upload.wikimedia.org/wikipedia/commons/2/24/Induced_fit_diagram.svg
Antibody – class of
proteins with common
structure: region
that is invariant and
region that varies a lot
(in different ab’s), the
latter having high, specific
affinity for some other
molecule (antigen, ligand)
Nature’s “professional
biosensor” molecule
Ball and stick model of crystal structure of portion of
antibody (left) binding protein from HIV (green, right).
Variable region of
antibody (purple)
Antibodies are most
common molecules
used to make
bio-assays specific
Antibodies to particular antigens can be generated in
animals, then made in large quantities in vitro
Base pairing –
at edges –
holds strands
together; each
bp = weak bond
3.3nm
10 bp (~1 kBT) but runs
of complementary
sequence ->
tight binding; can
be used for
specific recognition of NA’s with
2nm
compl. sequence
Nucleic acids – polymers of “bases”
DNA double helix
5
4
1
2
Biological Macromolecules - DNA
DNA double helix
3.3nm
10 bp
Base pairing –
at edges –
holds strands
together
Base stacking –
above & below compresses
ds into helix
5
4
Boiling separates
strands
1
2
2nm
RNA – like DNA, except OH at 2’ position, and Uridine for Thymine
Single-stranded (ss) nucleic acids (NA’s) often
used to detect complementary ssNA’s
because of incredible specificity
1 base mismatch can be detected in a 20 base long dna
How many different 20 base sequences are there?
420 = 1012
ss NA’s can also fold into shapes that bind other
molecules besides complementary NA’s
Aptamer = single
stranded nucleic
acid that happens
to have high
affinity for another
molecule
Aptamers can be
engineered and
selected for ability to
bind particular targets
Molecules used to provide specificity in biosensors
Enzymes – e.g. glucose oxidase for glucose
Antibodies
Genetically engineered antibody variants
Nucleic acids – hybridization
Aptamers – ss NAs that bind small molecules
natural and engineered
Fundamental relationship between NAs and proteins
Some protein enzymes move along DNA molecules
(molecular motors!), making RNA copy with
equivalent base sequence (“transcription”)
The RNA copy is then converted into a protein whose
amino acid sequence is determined by
the sequence of bases in the RNA (“translation”,
“genetic code”)
How do these motors work? How can they be studied?
= topics of later classes!
Immense medical significance
Variants in DNA sequence -> proteins with
variant amino acid sequence
Amino acid sequence determines how protein
folds, and hence its function
Engineered changes in DNA sequence -> novel
proteins, with possibly new functions
So big interest in sensors that determine DNA
sequence
While we will focus on biosensors (and a few
molecular motors), they are based on
the same interactions that occur naturally in
biological systems and hence provide
insight into biological systems
opportunity to develop innovative uses of
biological materials
opportunity to apply engineering tools
to better understand how biological
systems work
Approach – qualitative understanding of biosensor
phenomena, then quantitative analysis
Proto-typical biosensor – ELISA
Enzyme-linked immunosorbant assay
3. Add detection antibody that binds different site on target, wash
4. Detection antibody may be directly
attached to an enzyme (e.g. HRP)
that converts a substrate dye to a
colored molecule, or the enzyme
can be added on a 3rd molecule that
binds the detection antibody
5. Wash away enzyme not specifically attached
“receptor”
6. Add substrate and measure
color change
Typical ELISA format
1. Capture antibody (“receptor”)
usually immobilized on surface, e.g.
plastic 96 well (“mircrotiter “) plate
2. Test sample, that may contain target antigen (= analyte,
ligand), is added to well; target molecule sticks to capture antibody;
wash away whatever doesn’t stick
Typical protocol
Add sample in ~200ml, incubate ~1.5h (why so long?), wash
Add 20 Ab coupled to enzyme (e.g HRP).incubate 1.5h, wash
Add enz. substrate (e.g. tetramethylbenzene)
Incubate 30min (in dark)
Add stop solution (H2SO4) (why?), read OD (within 30min)
Analyte with know concentration serially diluted in some
wells to compare intensities
to that of test sample
Result: analyte conc. in sample
Many other assays are variants on this
with different “transducing” methods
e.g. fluorescence instead of dye color,
measure mass of attached molecules
instead of enzyme activity
measure electrical effects of captured complex
What determines sensitivity, incubation times?
How can we measure binding strength to target
vs other molecules in sample (-> false positives)?
Next few classes will develop simple binding kinetics model
to answer these questions
Reaction (receptor binding) kinetics
Let bm = total receptor conc. on sensor surface [moles/area]
b(t) = conc of receptors that have bound analyte at time t
Assume analyte binds receptor at rate ~
free analyte conc., c0,* free receptor conc., [bm – b(t)]
and dissociates from receptor at rate ~ b(t)
db(t)/dt = kon c0 [bm – b(t)] – koff b(t)
kon and koff are proportionality constants
db(t)/dt = kon c0 [bm – b(t)] – koff b(t)
Interpretation of binding constants
kon = av. # “binding” collisions per sec each receptor
molecule makes with an analyte molecule when analyte
conc = 1 in whatever units you use, e.g. #/m3 or “molar”, M,
moles/l
Units of kon are #/conc.*time, e.g. M-1s-1
koff = rate each receptor-analyte complex dissociates in #/s
Define KD=koff/kon Units of KD are conc., e.g. M
db(t)/dt = kon c0 [bm – b(t)] – koff b(t)
At steady-state, d/dt (b(t)) = 0, so
kon c0 [bm – b(t)] = koff b(t) =>
b(t)/bm = c0/KD /(1 + c0/KD)]
LHS = fraction of receptors that have bound target
Note it is natural to measure concentration of
free target molecules in units of KD
(unit check: are units of KD concentration?)
b(t)/bm = c0/KD /(1 + c0/KD)]
at steady state
If
c0 = KD, half of receptors have bound analyte
c0 >> KD, fraction of receptors with analyte -> 1
c0 << KD, fraction of receptors with analyte ~ c0/KD
i.e. most receptors are unoccupied
db(t)/dt = kon c0 [bm – b(t)] – koff b(t)
More generally, if c0 considered constant (often not true!),
b(t)/bm = fraction of receptors with analyte = A(1-e-Bt)
where A = [c0/KD /(1 + c0/KD)] and B = konc0 + koff
A = c0/KD /(1 + c0/KD)
b(t)/bm
Note exponential
approach to equil.
with characteristic
time t
time
t = 1/B = koff-1/(1+c0/KD)
c0/KD /(1 + c0/KD)
b(t)/bm
t = koff-1/(1 + c0 /KD)
typical values
time
kon ~ 106/Ms ( =10-21m3/s) fairly constant
koff ~ 1/s to 1/103s (varies a lot)
KD ~ mM (weak) to nM (tight binding)
Note smaller KD <-> tighter binding (slower koff)
There are many caveats to this model,
but it provides a simple way to begin
to evaluate systems quantitatively
The reasoning is completely general to other
biochemical interactions
Begin to think in terms of KD’s as natural
measures of strength of interactions
Main points:
Biological molecules are often polymers of simpler
subunits
They interact by standard laws of physics but
because their surfaces are highly variable (in
charge, dipolarity, other weak interactions)
they interact with each other in highly
“molecule-specific” ways
These interactions are often ~kBT so that complexes
form and dissociate at room temperature