Sample Data Sample Data 24.95 ut 0 15 30 45 60 75 90 105 - - PowerPoint PPT Presentation

Protein Adsorption

rotein Adsorption

• n
• n Solid Surfaces

Solid Surfaces

Chemical Engineering and Materials

Kim-Lien Thi Dinh

Major: Biochemistry Pasadena City College

Mentor: Kenny Rosenberg

Faculty Advisor: Jacob Israelachvili University of California, Santa Barbara

• Fundamental: Understand the interactions of protein

– Protein-protein interactions – Surface-protein interactions

• How much protein is on the surface?
• What would happen if…?
• Extend the life span of a biomedical implant

– Implant rejected by the body due to protein adsorption – Decrease the adsorption of protein

• Funding: National Institute of Health

– Improve people’s health – Prevent diseases/find causes and cures for diseases

Research and Motivation

esearch and Motivation

Surface-Protein Interactions

urface-Protein Interactions

• Proteins

– BSA (bovine serum albumin) – Tau

• Need a “balance”

– Quartz Crystal Microbalance (QCM)

• Super-sensitive balance
• Detects the mass of adsorbed protein
• Surfaces

– Gold – Mica

• Glue it on the crystal
• Make sure it works

How much protein is on the surface?

balance

0.0000 g

balance

1.0000 g

balance

0.0000 g

balance

3.0000 g

Q-Sense D300

• Sense D300

q-sense.com/Q-Sense D300 User Manual

14,999,900.00 Hz

• 100 Hz

15,000,000.00 Hz

(15 MHz)

14,999,950.00 Hz

• 50 Hz

15,000,000.00 Hz

(15 MHz)

Sample Data Sample Data

5 mg/ml BSA with 200 mM NaCl (15 Mhz)

• 120
• 100
• 80
• 60
• 40
• 20

20 15 30 45 60 75 90 105 Time (minutes)

∆ F req u en cy (H z )

How much BSA protein is

• n the gold surface?

24.95 25.05 25.15 15 30 45 60 75 90 105 Time (minutes) Temp(Celcius)

• 0.4

0.4 0.8 1.2 15 30 45 60 75 90 105 Time (minutes)

Dissipation X 1E-6

• 80 Hz ≈ 22 ng
• 95 Hz ≈ 26 ng

Addition of BSA

Dissipation

Sum of all energy losses in the system per

• scillation cycle

Soft/rigid film 1 monolayer of BSA ≈ 15 ng

0 µm 1 µm 0.5 µm 1 µm 0.5 µm 0 µm 1 µm 0.5 µm 1 µm 0.5 µm

Gold vs. Mica

0 nm 10 nm 5 nm 0.25 µm

GOLD

AFM

0.25 µm

MICA

AFM

0 nm 3 nm 2 nm

Preliminaries: 1mg/ml BSA on Mica

2.5 µm 0 µm 0 nm 2 nm 1 nm 0.5 µm

AFM

pH 10.15

2.5 µm 0 µm

Does the pH of the solution matter?

0 nm 2 nm 1 nm 0.5 µm

AFM

pH 6.80

0 µm 2.5 µm

pH 4.77

0 nm 5 nm 2.5 nm 0.5 µm

AFM

Preliminaries: Tau on Mica

Does Tau concentration matter?

0 nm 2 nm 1 nm 0 nm 2 nm 1 nm 0.25 µm

AFM

0.25 µm

AFM

1mg/ml BSA on Gold

Does the pH of the solution matter?

BSA on Gold BSA on Gold

Does the pH of the solution matter?

1 monolayer of BSA ≈ 15 ng

• 170 Hz ≈ 48 ng (3 layers)
• 13 Hz ≈ 3.5 ng

1mg/ml BSA with Acetic Buffer, pH 4.77

• 200
• 160
• 120
• 80
• 40

40 10 20 30 40 50 60 70 80 90 100 Time (minutes) ∆ Frequency (Hz)

1 mg/ml BSA with Carbonate Buffer pH 10.15

• 20
• 15
• 10
• 5

5 50 100 150 200 250 Time (minutes) ∆ Frequency (Hz)

First rinse Addition of BSA First rinse Addition of BSA

BSA on Gold BSA on Gold

Does salt concentration matter?

1 monolayer of BSA ≈ 15 ng

• 80 Hz ≈ 22 ng
• 300 Hz ≈ 83 ng (5.5 layers)

1 mg/ml BSA with K-PIPES, 1000mM NaNO3, pH 6.80

• 400
• 300
• 200
• 100

100 5 10 15 20 25 30 Time (minutes) ∆ Frequency (H z)

1 mg/ml BSA with K-PIPES, 1mM NaNO3, pH 6.8

• 100
• 80
• 60
• 40
• 20

20 5 10 15 20 25 30 Time (minutes) ∆ Frequency (Hz)

First rinse Second rinse Addition of BSA Addition of BSA First rinse

• It takes less than 5 seconds for the majority of

the protein to adsorb onto the surface

• pH of the solution matters
• Salt concentration matters too
• Produce a mica-modified crystal that works
• Study Tau

Summary and Future Research

ummary and Future Research

Acknowledgements

cknowledgements

• INSET

– Trevor Hirst –

• Dr. Nick Arnold

– Liu-Yen Kramer – Mike Northen

• Stu Feinstein
• UCSB, CNSI, PCC, ME

KENNY ROSENBERG Jacob Israelachvili and friends