MAE 6291 Biosensors and Bionanotechnology

Format lecture, discussion, lots of questionswill 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) detectedmolecules used to provide specificitytransducing modalities (light, mass, electricity)assay formats (sandwich, labels, label-free)processes affecting time to get signal (diffusion,

binding kinetics) and sensitivitymultiplex methods (e.g. hybridization arrays)massively parallel DNA sequencing methodsclinical 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, likemolecular motors, transduce chemical energy into motion

4. Appreciate overlap between engineering and biology

5. Gain experience reading research papers critically

Contact info: jesilver@gwu.edu, tel 240 447 3268set 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

ionssmall molecules (MW < 600g/mole=10-21g,

or ~50 atoms – e.g. glucose)peptides – short string of amino acidsproteins – string(s) of up to ~1000 amino acidsviruses - ~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, 1-100mMunits: 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)

What is significance of glucose in biology/medicine?

Diabetes – does it go up or down?problems if it goes upproblems if it goes down

H, O, C = hydrogen, oxygen,carbon atoms, etc.

Vertices = C atoms (understood)

Lines = covalent bondsstrength ~eV (1.6x10-19J)

More on units

Molecular weight = weight of NA (6x1023) molecules (=1 mole) in grams

H has molecular weight =1g/moleC “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 energyto break a covalent bond?

p ~ exp(-40kBT/kBT) = 10-18

So are covalent bonds usually stable at room temp.?

Another class of smallmolecules

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 longMWs ~100,000 g/mole (aka “kiloDalton”, kDa)

Protein polymers “fold up” into fairly compact units~10nm, based on weak interactions betweenamino acids

Some proteins fairly rigid = “fixed” structureoften 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, etcgroups -> specific interactions with othermolecules

Note how different from usual physics – gazillionsof identical electrons interacting uniformly

Glucose oxidase ~ 600 aa protein enzyme that binds and oxidizes glucose. Ribbon model of its aa backbone, por-tions 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 chargedsurface regions (red -, blue +), and some drug moleculesin binding pockets. Note complexity of surface allowing complex interaction with other molecules

http://www.pnas.org/content/104/1/42/F6.expansion.html

Proteins can interact forming larger polymers (of polymers) –> structural elements likefibers of collagen or microtubules (~25nm indiameter, microns long)

Proteins also can act as enzymes, “catalyzing”chemical reactions that break and reformcovalent bonds

http://upload.wikimedia.org/wikipedia/commons/2/24/Induced_fit_diagram.svg

Antibody – class ofproteins with commonstructure: regionthat is invariant andregion that varies a lot(in different ab’s), thelatter having high, specificaffinity for some othermolecule (antigen, ligand)

Nature’s “professionalbiosensor” molecule

Ball and stick model of crystal structure of portion of antibody (left) binding protein from HIV (green, right).

Variable region ofantibody (purple)

Antibodies are most common moleculesused to make bio-assays specific

Antibodies to particular antigens can be generated inanimals, then made in large quantities in vitro

DNA double helix

2nm

3.3nm10 bp

12

45

Base pairing –at edges – holds strandstogether; eachbp = weak bond(~1 kBT) but runsof complementarysequence ->tight binding; canbe used for specific recogni- tion of NA’s withcompl. sequence

Nucleic acids – polymers of “bases”

DNA double helix

2nm

3.3nm10 bp

12

45

Biological Macromolecules - DNA

Base pairing –at edges – holds strandstogether

Base stacking –above & below -compressesds into helix

Boiling separatesstrands

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’sbecause of incredible specificity

1 base mismatch can be detected in a 20 base long dnaHow many different 20 base sequences are there?

420 = 1012

Aptamer = singlestranded nucleicacid that happens to have highaffinity for anothermolecule

Aptamers can beengineered and selected for ability tobind particular targets

ss NA’s can also fold into shapes that bind other molecules besides complementary NA’s

Molecules used to provide specificity in biosensors

Enzymes – e.g. glucose oxidase for glucoseAntibodiesGenetically engineered antibody variantsNucleic acids – hybridizationAptamers – 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 whoseamino acid sequence is determined bythe 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 withvariant amino acid sequence

Amino acid sequence determines how proteinfolds, and hence its function

Engineered changes in DNA sequence -> novelproteins, with possibly new functions

So big interest in sensors that determine DNAsequence

While we will focus on biosensors (and a fewmolecular motors), they are based onthe same interactions that occur naturally inbiological systems and hence provide

insight into biological systemsopportunity to develop innovative uses of

biological materialsopportunity to apply engineering tools

to better understand how biologicalsystems work

Approach – qualitative understanding of biosensorphenomena, then quantitative analysis

Proto-typical biosensor – ELISA

Enzyme-linked immunosorbant assay

1. Capture antibody (“receptor”) usually immobilized on surface, e.g. plastic 96 well (“mircrotiter “) plate

3. Add detection antibody that binds different site on target, wash4. Detection antibody may be directly

attached to an enzyme (e.g. HRP)that converts a substrate dye to a colored molecule, or the enzymecan be added on a 3rd molecule thatbinds the detection antibody

5. Wash away enzyme not specifically attached6. Add substrate and measure

color change

“receptor”

Typical ELISA format

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?), washAdd 20 Ab coupled to enzyme (e.g HRP).incubate 1.5h, washAdd 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 thiswith different “transducing” methods

e.g. fluorescence instead of dye color,measure mass of attached molecules

instead of enzyme activitymeasure electrical effects of captured complex

What determines sensitivity, incubation times?

How can we measure binding strength to targetvs other molecules in sample (-> false positives)?

Next few classes will develop simple binding kinetics modelto 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 offree 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 analytec0 >> KD, fraction of receptors with analyte -> 1c0 << 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

b(t)/bm

time

A = c0/KD /(1 + c0/KD)

t = 1/B = koff-1/(1+c0/KD)

Note exponentialapproach to equil.with characteristictime t

b(t)/bm

time

c0/KD /(1 + c0/KD)

= t koff-1/(1 + c0 /KD)

typical values kon ~ 106/Ms ( =10-21m3/s) fairly constantkoff ~ 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 beginto evaluate systems quantitatively

The reasoning is completely general to otherbiochemical 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 butbecause 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