1. PittCon 2001: Paper #645 Mark G. Hartell and W. Charles Neely
NOVEL SENSOR DESIGN PLATFORM COMBINING MOLECULAR SELF-ASSEMBLY WITH LANGMUIR-BLODGETT TECHNOLOGY FOR THE ASSESSMENT OF UNIQUE CELL-SPECIFIC PEPTIDES
Mark G. Hartell and W. Charles Neely
Department of Chemistry
Alexander M. Samoylov
Tatiana I. Samolylova and Bruce F. Smith
Scott-Ritchey Research Center
Vitaly Vodyanoy
Department of Anatomy, Physiology and Pharmacology
Auburn University
MDA * DARPA * U.S. Army

2. Project Overview Project Background: Project Requirement:
Identify cell surface markers specific to muscle myotubes in the development of a novel class of gene therapy vectors to combat forms of muscular dystrophy
Project Requirement:
A rapid and simple test bed to measure specificity of selected peptide ligands against a variety of tissue homogenates
Novel Application: Langmuir-Blodgett and Self-Assembly
Design and construct a novel sensor using LB thin-film technology and molecular self-assembly to yield a peptide-specific sensor

3. Selection of Tissue-Specific Ligands using a Random Peptide-Presenting Phage Library
Phage Library Concept
Insertion of a DNA fragment, fixed in length but with unspecified codons, in a phage surface protein gene, pIII, results in a fusion protein on the phage surface
Phage surface protein gene expresses codons for specific peptides to attach to specific tissues in host
Isolating phage DNA yields sequence information for this tissue-specific peptide

4. The Langmuir-Blodgett Technique
Molecule of interest is spread into a monolayer film
Area dimensions are controlled by a movable barrier
Molecular orientation well controlled in 2-dimensions
Can be used to engineer highly ordered, molecularly thin layers

5. LB Film Deposition System
SYSTEM SUMMARY
System contained in laminar flow hood
Trough is milled from solid Teflon block
Computer controlled
KSV 2200 LB System
Deposition Parameters
20  0.1 C set = 25 mN/m
25 mm/min compression to set
Subphase pH 7.41
55 mM KCl, 4 mM NaCl, 0.1 mM CaCl2, 1 mM MgCl2 & 2 mM MOPS

6. LB Technology and Biosensor Construction
Determination of surface properties provides unique understanding of molecular orientation and monolayer stability
COME TO TALK ON FRIDAY
LB technology provides heightened control of compact, organized, molecularly thin films
Ultra-thin film biosensors yield competitive sensitivity and detection limits
Simple sample preparation and stability of fluorescence
Hartell, M.G., Neely, W.C., Vodyanoy, V. “Study of the Chemical, Physical and Optical Properties of a Lipophilic Hydroxycoumarin Analog in Mixed Monolayers.” Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. Abstract #1338. Seminar. New Orleans, LA, March 4-9, :50 AM FRIDAY
Pathirana, S.T., Barbaree, J., Chin, B.A., Hartell, M.G., Neely, W.C., Vodyanoy, V. Rapid and Sensitive Biosensor for Salmonella. Biosensors Bioelectron, 15, , 2000.

7. Quartz Crystal Microbalance
SPECIFICATIONS
Measures AT-cut planar quartz crystals with a 5 MHz nominal oscillating frequency
Frequency resolution of 0.5 Hz at 5 MHz
Mass resolution of 10 ng/cm2
Maxtek PM-700 Plating Monitor
QCM Methods
QCM crystals coated with sensor material
Homogenate tested in PBS
Test 1 mL ambient solution
Signal relative to PBS blank
m ~ V

8. Close-Up View of QCM Crystals
Probe Arm
Obverse
Reverse
Mass loading onto Au electrode surface changes measured oscillating frequency of quartz wafer

9. Why Use LB Technology?
QCM
YYYYYYYYYYYYY
YYYYYYYYYYYYY
QCM
QCM
YYYYYYYYYYYYY
YYYYYYYYYYYYY
QCM
QCM
Thin-film biosensors have the benefit of lower mass loading for increased detection sensitivity

10. Approach to Sensor Construction
Need a manner by which a peptide strand can be attached to the QCM substrate
LB technology provides a technique to produce molecularly thin, highly organized films – but depositing peptides is a problem
Can use LB technology to construct an organized foundation onto which the peptide may be attached
Attach peptide using a molecular self-assembly procedure
Combination of both techniques provides better control over sensor construction than either singular approach

11. LB Films and Molecular Self-Assembly
QCM Crystal: Peptide Sensor to Differentiate Tissue
Gravimetric detection of binding event
Sensor designed to be specific against mouse muscle tissue
Incorporates novel mix of LB film with molecular assembly
Make organized lipid foundation via LB technology
Assemble sensor onto organized foundation to yield organized complex design
General Design Concept
Goals:
Use sensor as test fixture in selecting tissue-specific ligands
Novel testing regime allows for fast determination of specificity and sensitivity of test ligands
Application aimed at discovering human-tissue ligands for better therapeutic drug designs

12. The Foundation: a Biotinated Phospholipid
First Step in Sensor Construction
Lipid tail is ideal for LB film construction
Phospholipid structure compresses to a stable, highly organized monolayer film
Film may be deposited successfully onto QCM surface
Biotinated headgroup provides a site for further attachment via molecular assembly

13. Position of QCM slides during deposition 2 sets back-to-back crystals
LB Film Deposition
DEPOSITION SUMMARY
QCM crystals placed back-to-back: deposit material on one side only
Compress biotinilated lipid to a constant 25 mN/m at 20 C
~9 layers deposited
Transfer ratios indicate Y type deposited layers
Position of QCM slides during deposition
2 sets back-to-back crystals
Transfer ratios during deposition similar to LB literature for biomolecules

14. The Glue to Hold Everything Together: Streptavidin
Streptavidin has high affinity for biotin
Streptavidin can bind two biotin molecules on each face
Provides the middle layer of this molecular assembly “sandwich”
Four binding sites are
symmetrically positioned
Step 3
Step 2
Step 1
add
streptavidin
add
biotin-protein
LB film

15. Peptide Ligand Found to be Selective to Mouse Muscle
Muscle Blocked Kidney Brain Liver
QCM Response Voltages
Muscle
Indicates stronger response with mouse muscle versus kidney, brain and liver
Sensitivity and Vmax
Clear difference in response characteristics with mouse muscle blocked by peptide

16. Blocking Studies Confirm Specificity
Tissue homogenate Kd, mg/mL Vmax, mV
Mouse muscle ± ± 4.0
Blocked m. muscle ± ± 3.0
Kd were calculated from Hill plots, Vmax were estimated from dose-response curves.

17. Conclusions
Mouse Tissue Sensitivity (S) Selectivity coef. (K) Selectivity ratio (R)
mV/decade Relative unitsa Relative unitsb
Muscle 134 ±
Kidney ± 7.0 (3.1 ± 1.0) × (9.3 ± 3.2) × 10-5
Brain ± 16 (4.6 ± 1.4) × (3.4 ± 1.1) × 10-3
Liver ± 6.0 (5.0 ± 1.5) × (1.1 ± 0.4) × 10-3
a Change of the output voltage is empirically described as: V = A + S log C,
C = bulk concentration of tissue homogenate, A = constant, S = slope of dose response dependence, defined as the sensitivity of the sensor.
Coef. selectivity defined as the ratio of slopes: K = Stissue/Smuscle
b Selectivity ratio (R) is defined as the concentration ratio of the primary to interfering tissue homogenate (Cmuscle/ Ctissue) which gives the same response change at the same conditions. R was calculated at 0.01 mg/mL of muscle homogenate using experimental equations:
 Vmuscle = log C  Vkidney = log C
 Vbrain = log C  Vliver = log C

18. Conclusions
LB technology may be used in conjunction with other techniques, such as molecular self-assembly to yield better biosensor construction
Ultra-thin film biosensors constructed from LB monolayers may be a fast and simple alternative to selectivity determinations