Microfluidic Immunoassays in Polymeric ChipsW. W. P. Chang 1, C. Li1, L. Bousse 1, T. Kawabata1, Y. Shih1, C. Kagebayashi2,

T. Kurosawa2, H. G. Wada1, M. Watanabe2, S. Satomura2

1Wako Pure Chemical Industries Ltd., Mountain View R&D Center, Mt. View, CA, USA2Wako Pure Chemical Industries Ltd., Osaka Research Laboratories, Amagasaki, Japan

Introduction

A quantitative method for performing rapid

immunoassays was recently developed utilizing

microfluidic quartz chips1. Here we demonstrate

the performance of a diagnostic application (alpha-

fetoprotein AFP-L3 or AFP-L3 assay) on

disposable thermoplastic chips.

AFP-L3 is an isoform of AFP. AFP-L3 is a

biomarker specific for liver cancer (HCC)2. The

ratio of AFP-L3 to total AFP, or %L3, has been

established as a sensitive marker for patients at

risk for HCC3.

Isotacho-phoresis (ITP) and capillary gel

electrophoresis (CGE) in polymer chips are used

to react the sample and reagents, and to separate

AFP-L3 immunocomplex within 120 s. The

sandwich immunoassay using the electrokinetic

analyte transport method (EATA) yields high

sensitivity, good reproducibility and linearity.

References1. T. Kawabata et al., Electrophoresis (2008) 29: 1399-1406.2. K. Taketa et al., Gastroenterology (1990) 99:508-518.3. H. Oka et al., J Gastroenterol Hepatol (2001) 16:1378-1383.4. C. Park et al., Anal Chem (2008) 80:808-814.

Conclusions

� Disposable microfluidics plastic chips can be

used for rapid diagnostics applications, such as

AFP-L3 assay

� Data is quantitative, linear and reproducible

� Sensitivity is excellent. LOD is 0.014ng/mL

(0.2pM) for AFP

Results and DiscussionA known concentration of purified AFP-L1 and

AFP-L3 is mixed in a BisTris sample buffer to form

the sample. Two monoclonal antibodies

recognizing different epitopes on AFP are

conjugated to either DNA or fluorescent dye.

The DNA tag enhances mobility of the immuno-

complex. LCA (Lens culinaris agglutinin) binds the

alpha-1,6-fucosyl residue (core fucose) unique to

AFP-L3 isoform but not AFP-L1, which shares the

primary sequence. Such binding allows AFP-L3 to

separate from AFP-L1.

DNA-mAb, sample and dye-mAb are loaded into

predetermined zones on microchip (see Scheme),

then high voltage is applied across the EATA zone

for ITP stacking and immunocomplex formation.

AFP-mAb complexes are further separated into

AFP-L1 and AFP-L3 subgroups through LCA

binding after the stacked material enters the CGE

zone.

Contact Information

William W. P. Chang, Ph. D.

w-chang@wakousa.com

650-210-9153 x116

L1mAb1 mAb2DNA- - - - -

- - - - -

L3mAb1

α-1,6-fuc

mAb2DNA- - - - -

- - - - -

DNA-mAb conjugate 1 enhances mobility towards the anode; fluorescentdye-mAb conjugate 2 allows detection of complex, while LCA effects theseparation of AFP-L1 and -L3 isoforms (glycoforms)

AFP-L3 Sandwich Immunoassay

Using the AFP-L3% sandwich immunoassay on

disposable, plastic microfluidic chips, we

observed

1. Good resolution of AFP-L1 and AFP-L3 (Fig. 1);

2. Results that are quantitative and reproducible

(Table 1);

3. Excellent linearity and specificity (Fig. 2 & 3);

4. Limit of detection for AFP-L1 is 0.014 ng/mL

(based on 3SD of noise, data not shown);

5. Good assay sensitivity: 2% AFP-L3 is detected

in 25 ng/mL of total AFP (Fig. 4);

6. Fast analysis: assay completed within 120s

after field is applied.

We are currently developing an automated

instrument for this assay using plastic

microchips.

cancerous

benign

5 Assay Dynamic Range

y = 2.5463x + 55.803

R2 = 0.9996

0.0

1000.0

2000.0

3000.0

0 300 600 900 1200

AFP L1 (ng/mL)

L1 P

eak A

rea

Figure 5. AFP assay dynamic range; inset shows a

lower concentration range.

Material and Methods

4 AFP-L3% Sensitivity

Figure 4. AFP-L 3% sensitivity. 2% AFP-L3 is

detectable at 25ng/mL total AFP (L1+L3). At 10ng/mL

total AFP, as low as 5% AFP-L3 can also be detected

(data not shown).

15

17

19

21

23

25

55 57 59 61 63 65

Time (s)

RF

U

0% L3

2% L3L1

L3

AFP-L1 Linearity2

Figure 2. Relationship between AFP-L1 concentration

and AFP-L1 peak height.

y = 3.272x + 53.455

R2 = 0.998

0

400

800

1200

1600

2000

2400

2800

3200

3600

0 400 800 1200

AFP- L1 Concentration (ng/ mL)

AF

P-

L1

Pe

ak H

eig

ht

(RF

U)

AFP-L3% Linearity and Specificity

Figure 3. Known ratios of purified AFP-L1 and -L3

were applied. Relationship between the measured

AFP-L3 % and expected values are shown.

3

y = 1.0004x - 0.0235

R

2

= 0.9997

0.0

20.0

40.0

60.0

80.0

100.0

0 20 40 60 80 100

Theoritical AFP- L3%

Exp

eri

me

nta

l A

FP

-L

3%

Assay Reproducibility

Table 1. Reproducibility (CV) of migration time, quantifi

-cation, resolution and AFP-L3 ratio (N=10).

N=10 L1 Time(s) L3 Time(s) L1 Area L3 Area L3% Resolution

CV 0.97% 0.96% 1.79% 3.04% 0.96% 4.96%

ITP-CE Electrophoregram1

Figure 1. An example of AFP-L1 and -L3 separation

(500pM, AFP-L3 is 50% of total AFP).

280

330

380

430

480

530

580

630

55 60 65 70

Time (s)

RF

U

AFP-L1 AFP-L3

TB:trailing buffer

LB: leading buffer

HO:floating electrode (“hand-

off”)

EATA: incubation &

concentration

Dashed arrow shows the transition from cathode to floating electrode, after ITP stacking (“hand-off”). A field is then applied between HO and anode for CGE separation4

TB LB

HO

floating electrode

CGEstackingEATA

anodecathode

LIF detector

Sample +Dye-mAbDNA-mAb

TB LB

HO

floating electrode

CGEstackingEATA

anodecathode

LIF detector

Sample +Dye-mAbDNA-mAb

Microchannel Scheme

0.00

500.00

1000.00

1500.00

2000.00

0 50 100 150 200 250

AFP L1 (ng/mL)

Wako