MAE 6291 Bionanotechnology and Biosensors

Goals:1. learn about nanotechnology-based biosensors

molecules (analytes) detectedmolecules used to provide specificitytransducing modalities (light, mass, electricity)assay formats (label-free, sandwich, labels)processes affecting time to get signal and sensitivity

(analyte diffusion, binding kinetics)multiplex methods (e.g. hybridization arrays)massively parallel DNA sequencing methodsbiological significance of assays

2. At end of course, be able to design assay for 1 or moreanalytes using different modalities,predict sensitivity, specificity, describe expected technical challenges

3. Have framework for considering clinical utility:how well do results correlate with medical state?is concentration or presence/absence critical?will assay fill need or create problems?

4. Gain experience reading papers in field critically

Hopefully, good intro/entrée into world of molec. biol. for engineers, should be growth sector (!)

Format - lecture discussion

% gradewill aim to have students present

segments of papers in each class .25

homework ~1 every 1 - 2 classes to learnhow to use what we cover .25

will try to include demonstrations – e.g. ELISA,fluorescence microscopy, gene chip, pcr

take-home midterm exam .25

student presentation/or take-home final exam .25

Papers, lecture notes, homework, previous week’s homework answers, announcementswill be on Blackboard

Contact info – Prof. Jon SilverPhillips Hall 738 jesilver@gwu.edu, cell 240 893 7020

What makes something a “bio” sensor?

target molecule is biological

molecule used to provide recognition specificity is biol.(enzyme, antibody, aptamer)

analog of biol. process contributes to sensor designe.g. evolution/selection for improved functionality

design mimics biological organ – e.g. compound eye

Molecules (things) to be detected

ions – e.g. Na+

small molecules (MW < 600g/mole=10-21g, or ~50 atoms – e.g. glucose)

peptides – short string of amino acidsoligonucleotides – short string of nucleic acids

= bases A, G, C, T (U) – joined via sugar-PO4

proteins – string(s) of up to ~1000 amino acidsviruses - ~1000+ proteins + NA genome (>104 bases)larger organisms – bacteria, protists, cellsnucleic acid sequence

Protein = linear polymer of amino acids (aa)

chains from a few to ~1000 aa long

aa order encoded in order of bases in DNA

order of aa’s determines protein’s structure, interacting surfaces, properties, function

All NH2-CHX-COOH side groups X differ

hydrophobic chains hydrophobic rings polar, not charged + charge at neut pH - charge at neut pH

give proteins highly variable chemcial surfaces for specific identification and inter-action with other molecules

Model of transmembrane 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

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”

Cheap to make mmol of DNA chains with arbitrary seq. up to ~100 bases long for specific sensing elements (<1$/base)

Molecules used to provide specificity

Enzymes – e.g. glucose oxidaseAntibodiesNucleic acids – hybridizationAptamers – ss NAs that bind small molecules

natural and engineeredAntibody variants and substitutes

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

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

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

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

Assay formatsbulk solution (e.g. signal generated by molecules

coming together on DNA)surface sensors (the majority)

captured analyte -> signal directlye.g. due to mass, D index of refraction

sandwich – capture analyte, then add labeledmolecule/particle that binds analyte

label provides enhanced signal – e.g.radio-isotopefluorescenceinc. mass (e.g. gold beads)enzyme on second antibody can generate multiple signal mol. dyes or chemi-luminescence = signal amp.

More assay formats“homogeneous” assays (no washing needed)“coincidence” – require 2 or more specific binding

events (e.g. sandwich, increases specificity)massively parallel hybridization arrays: different DNA

species in each position

DNA synthesized in situ DNA attached to micron-sizedvia photo-lithography beads in wells etched in silicon

Specialized processes/formats

target amplification (rather than signal amplification)NA targets can be copied enzymatically

(pcr, polymerase chain reaction) to yield~109 replicates before detection

massively parallel DNA sequencing in arrays of wells, each containing many copies

of a different dna fragment made by pcrin DNA “thickets”, each containing many copies

of a different dna fragment grown on glass by pcr

Signal transduction methods

light – colorimetry (dyes), luminescence, fluorescence, fl.res. energy transfer (FRET-sensitive to nm separation)evanescent wave effects to reduce bkgdsurface plasmon resonance (SPR)

electrochemical – oxidation/reduction rxns on surface transfer electrons to/from ions in solution -> currentalters V-I relations, often transientlye.g. glucose oxidase sensors

electrical – field effect transistors (FETs) nearby charge affects V-I relation

ion sensitive-FETS used in new dna sequencing meth.carbon nanotube FETs

Transduction methods - mechanical

micro/nano cantilevers, analyte binding changes mass -> D in resonance freq. electrical or optical read-out

DNA tethering micron-sized beadsbeads visualized microscopically, binding molecules alter tether properties -> new kind of single-molecule sensors

Goals increased sensitivityincreased parallelization

Lots of room for innovationminiaturizationcost reductionuse of new nanoscale phenomena

Clinical Utility – what is it useful to detect?

Infectious disease agents – e.g. viruses whose presence always indicates clinically significant infection or contamination – HIV, HBV, HCV, polio, malaria

But other infectious agents are normally present inenvironment, so detection may or may notbe clinically significant – e.g. streptococci

Proteins absolutely diagnostic of cancer – e.g. fusion protein (bcl) that only occursin chronic myelogenous leukemia(a result of a chromosomal translocation)

But this is exception: most proteins are normallypresent; their concentrations may changein disease but often they change in manyconditions, so changes are not diagnostic,though possibly suggestive

Our ability to detect things is outstripping ourability to know what to do with the results

Example – prostate specific antigen (psa)

serum level elevated (>4ng/ml) in blood of menwith prostate cancer, but also in men withprostate inflammation

not elevated in all men with prostate cancer(false negatives); elevated in some menwithout any disease (false positives)

another problem – overdiagnosismany men with prostate cancer detected by PSAand biopsy (bx) have such slow growing disease they would never have symptoms and dye of

something else; elevated PSA -> medical testsand procedures (bx, surgery) that often have severe side-effects, sometimes providing no benefit

After >10 years of PSA testing, clinical trials with > 100,000 men showed PSA screening ->increased diagnosis (expected) but noimproved survival

Other quandaries:

Genetic tests can identify people with increased riskof senile dementia for which no preventive measures are known

Genetic tests can identify people with increased riskof some cancers for which we have no effectivescreening tests (ovarian cancer)

Some new tests identify patterns of altered protein levels or genetic changes in patients with breast cancer that are reported to correlate with worse prognosis-> altered chemotherapy

The correlations between panels of “biomarkers”and clinical state result from data-mining studieswhich are subject to statistical pitfalls – e.g.large # of possible patterns increase chance that some pattern will correlate with outcomein any finite study - but won’t be reproducible

Implication – need to be cautious about over-estimatingclinical value of diagnostic tests made possibleby new technology, esp. given escalating costs

Processes affecting time to detect analyte and sensitivity(subject of next 2 classes)

Binding kinetics – of analyte to sensormass action drives bindingconcentrations of analyte and capture probe

very importantoften limit sensitivity

How does analyte get to capture molecule?diffusion (usually on small scales): t~x2 (not x)result of random (Brownian) collisionsfast over short distances (nm), slow over long

(mm); scale determined by D (diff. const.)

Flow (advection) – often used to introduce sample, labelinto sensor, wash out non-binding proteins

Competition between advection and diffusion: narrow sensor channel reduces time for analyte to

diffuse to sensor surface but also reduces amount of sample that can be introduced and increases viscous drag

flow replenishes analyte depleted from regionnear to sensor (so speeds up binding)but if too fast, analyte molecules leave chamber before they can bind

balance between flow rate and diffusion rateoptimizes performance but sets limitsto how fast device can function