Eletrokinetic Protein Preconcentration Using Nanochannels Formed By - - PowerPoint PPT Presentation

Eletrokinetic Protein Preconcentration Using Nanochannels Formed By Weak Bonding of PDMS Membrane with Glass Substrate

Sun Min Kim

• Dept. of Mechanical Engineering,

Inha University

• 2008. 4. 18.

University of Michigan, Ann Arbor Department of Mechanical Engineering

INHA University Department of Mechanical Engineering

The 5 The 5th

th Korea

Korea-

• U.S. Nano Forum

U.S. Nano Forum

• 2-

INHA University Department of Mechanical Engineering

Bio-Micro Fluidics Lab.

Lab on a chip (Laboratory on a chip)

Why concentrate samples?

– Sensitivity: Concentration in sample often less than detection limits of instruments – Waste: Most samples are > 1 mL in size, but only nanoliters can be analyzed at a time on a microchip

Lab on a Chip

Sample acquisition Sample preparation Analyte detection

Analyte Concentration Analyte Amplification

• 3-

INHA University Department of Mechanical Engineering

Bio-Micro Fluidics Lab.

PDMS Glass

PBS + Protein sample

PBS Buffer

Gap:20μm

+ HV 2 1 4 3 5

Separation channel

GND PDMS Glass

PBS + Protein sample

PBS Buffer

Gap:20μm

+ HV + HV 2 1 4 3 5

Separation channel

GND

M M’

PDMS Glass

PBS + Protein sample

PBS Buffer

Gap:20μm

+ HV 2 1 4 3 5

Separation channel

GND PDMS Glass

PBS + Protein sample

PBS Buffer

Gap:20μm

+ HV + HV 2 1 4 3 5

Separation channel

GND

M M’

PDMS Glass

PBS + Protein sample

PBS Buffer

Gap:20μm

+ HV 2 1 4 3 5

Separation channel

GND PDMS Glass

PBS + Protein sample

PBS Buffer

Gap:20μm

+ HV + HV 2 1 4 3 5

Separation channel

GND

M M’

Simple Glass/PDMS Preconcentrator

• Channel dimensions

Chevron channels (W 40µm× D 18µm, L=16mm), Separation channel (W 30µm× D 18µm, L=40mm)

• Microchannels are created by casting PDMS over a mold

(SU-8 on a Si substrate).

–

+

PDMS Glass M M’ 20µm

• 4-

INHA University Department of Mechanical Engineering

Bio-Micro Fluidics Lab.

Experimental Results

Experimental conditions

• Protein sample : Fluorescein isothiocyanate (FITC) conjugate bovine

serum albumin (BSA) and ovalbumin (OVA)

• Buffer : Phosphate-buffered saline buffer (10mM, pH 7.4) and

Phosphate buffer (20mM, pH 7.2)

• O2 plasma treatment of the PDMS (100W, 200mTorr, 3min)
• Applied electric potential: 100, 200, 300V

PDMS Glass

PBS + Protein sample

PBS Buffer

Gap:20μm

+ HV 2 1 4 3 5

Separation channel

GND PDMS Glass

PBS + Protein sample

PBS Buffer

Gap:20μm

+ HV + HV 2 1 4 3 5

Separation channel

GND

100µm 100µm 100µm

• 5-

INHA University Department of Mechanical Engineering

Bio-Micro Fluidics Lab.

Experimental Results (cont’d)

Concentration of FITC-BSA in the preconcentration zone

Preconcentration Zone

500 1000 1500 2000 2500 3000 5 10 15 20 25 30 35 1pM 100pM 10nM 5µM standard 10µM standard 20µM standard Fluorescence Intensity (A.U.) Time (min)

Concentration achieved up to 106 –fold

• 6-

INHA University Department of Mechanical Engineering

Bio-Micro Fluidics Lab.

Separation of preconcentrated proteins

• 7-

INHA University Department of Mechanical Engineering

Bio-Micro Fluidics Lab.

Physical Mechanism: Hypothesis and Verifications

“nanoscale channels” formed between the PDMS and the glass due to the weak, reversible bonding

• 1. No preconcentration occurs in PDMS/PDMS and irreversibly bonded PDMS/glass

(stronger bond) chips.

• 2. Nanochannels work as a cationic selective membrane due to the Ion exclusion-

enrichment effect (EEE) caused by electrical double layer (EDL) overlapping.

• 3. Electroosmotic flow (EOF) dominates over electrophoresis (EP)

2 1 4 3 5

M M’

2 1 4 3 5

M M’ M M’

M M’

PDMS Glass

Ⓘ

Hm Hn – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕ ⊕

λD

⊕⊕⊕⊕⊕⊕⊕⊕ ⊕ ⊕ ⊕⊕⊕⊕⊕⊕⊕⊕ ⊕⊕⊕⊕⊕⊕⊕⊕ ⊕⊕⊕⊕⊕⊕⊕⊕ ⊕ ⊕

Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ Ⓘ

EOF EP

–

+

• 8-

INHA University Department of Mechanical Engineering

Bio-Micro Fluidics Lab.

Experimental Verifications

Verification of the cationic selectivity of the nanochannel

– Sample: 1µM Rhodamine 123 dye (cationic) in PBS buffer (bottom)

• 9-

INHA University Department of Mechanical Engineering

Bio-Micro Fluidics Lab.

Experimental Verifications (cont’d)

Verification of the dominant electroosmotic flow (EOF)

– Sample: 1µM FITC-BSA in PBS buffer (Top)

• 10-

INHA University Department of Mechanical Engineering

Bio-Micro Fluidics Lab.

Conclusions

• PDMS / Glass chip for protein preconcentration

was designed and fabricated.

• Preconcentration of labeled BSA (FITC BSA) has

been achieved up to 106 – fold.

• Preconcentrated protein was injected and

separated in a separation column.

• “Nanoscale channels” formed between the PDMS

and the glass due to the weak, reversible bonding works as a cationic selectivity membrane by Ion exclusion-enrichment effect (EEE).