Biosensor for Point-of-care Testing

Biosensor
for
Point-of-care Testing
Biodevice Technology, Ltd., Japan

� About us
� An enhancement assay for
immunochromatographic test
� Disposable printed electrodes for biosensors,
DEP-Chips, and their applications
� immunosensor, GLEIA
� Genosensor
� Conclusion

BioDevice Technology, Ltd.
BDT is a company born from Japan Advanced Institute of Science
and Technology (JAIST) and was established in July 2003 by
Professor Eiichi Tamiya and five faculties.
Capital 2 million yen, Employees 6
Our Objects of Establishment
・Establish products as fruits of research in
universities.
・Back up of making collaboration academic
researchers with companies.
・Build innovation by collaboration among
academic, companies and government
R & D of Biosensors
・High Sensitive Immunochromatography
(PCT/JP2006/323617)
・DEP-Chip series for electrochemical biosensors
Gold-Linked Electrochemical Immuno Assay
(GLEIA, PCT/JP2007/56992)

Applications
Immunochromatographic Test Strip and Applications
Clinical Diagnosis
Pregnancy, cancer, infectious disease,
cardiovascular problems etc.
Food safety test
Allergens, food toxins, GMO
Ecological detection
Endocrine disturbing chemicals
Marketplace in Japan
(2006)
$250 million
Influenza diagnosis kits
in Japan
$110 million
Advantage:
•User friendly format, easy to use
•Rapid to obtain test result
•Less interface due to chromatographic separation
•Relative low cost
Problem:
• Relative low sensitivity around 1 ng/mL
• Semi-quantitative
Suit with screening or POC tests

Enhancement Assay for Immunochromatographic test strip
antigen
Nanogold labeled antibody
Apply sample solution to a
conjugation pad
Conjugation pad
Test line
Control line
Antigens react with nanogold- Antigen-antibody
labeled antibodies and
complex
complex flow to capture antibodies
Capture antibody on test line
Antibody for control line
(PCT/JP2006/323617)
flow
Absorption pad
Normal strip Enhancer-based strip
The complex of antigen and nanogold labeled antibodies accumulated and a higher amount of
gold nanoparticles in close proximity were brought in the enhanced test strip. As a result, higher
Intensity of the localized surface plasmon resonance absorbance of the nanogold particles are
able to obtain and a distinct red color can be easily observed.

Enhancement Assay for Immunochromatographic test strip
Normal and the enhancer-based strips for the detection of hCG in PBS
Normal strip Enhancer-based strip
1000 10 Control 1000 10 Control
100 1 100 1
(pg/ml) (pg/ml)
Control line
Test line
Intensity
50
40
30
20
10
0
Normal 従来型ストリップ strip
Enhancer-based strip
金ナノ粒子固定化
トリップ(1
1000 100 10 1 Control
Concentration of hCG (pg/ml)
In PBS
The lower limit of detection was 100pg/ml
in the normal strip, whereas it was 1pg/ml
in the enhancer-based strip.
In serum
The lower limit of detection was 100pg/ml
in the normal strip, whereas it was 10pg/ml
in the enhancer-based strip.

Our Applications
Object Species Significance
PSA human Prostate cancer marker 2 ng/mL
hCG human Pregnancy maker 1 pg/mL
Lower limit of
detection
Insulin human Metabolic syndrome marker 125 pg/mL
Hemoglobin human Gum disease marker 30 ng/mL
HbA1c human Blood glucose level marker 0.25 ng/mL
Ig E human
Maker of allergy to cedar
pollens, mite and cat hair
Level 2
sIgA canine Stress marker 20 ng/mL
Cortisol human Stress marker 8 ng/mL
Testosterone human Climacteric marker 125 pg/mL
E. coli E. coli Microbial contamination OD600 0.3

Disposable Type Screen Printed Electrodes
DEP-Chip Series
Square working electrode
SP-N, SP-P
(carbon)
SR-N
(gold)
Round working electrode
Trial manufacturing of originally-designed
screen printed electrodes
EP-N
(carbon)
EP-PP
(carbon, WE with ring and dam)
ER-N
(gold)

Cyclic voltammograms at 0.1 V/s (WE: carbon)
3 mm
Red line: 2 mM K3[Fe(CN)6] + 0.3 M Na2SO4
Blue line: 0.3 M Na2SO4
11 mm
Ipa (anodic): 10.8 μA
Epa (anodic): 0.163 V
DEp:0.124 V
3 mm
Ipa (anodic): 10.8 μA
Epa (anodic): 0.166 V
DEp:0.141 V
11 mm

Cyclic voltammograms at 0.1 V/s (WE: gold)
Red line: 2 mM K3[Fe(CN)6] + 0.3 M Na2SO4
Blue line: 0.3 M Na2SO4
3 mm
11 mm
Ipa (anodic): 15.8 μA
Epa (anodic): 0.107 V
DEp:0.066 V

• Disposable
Advantage of DEP-Chips
• No contamination
• Easy to use
• Low cost
• High quality CV < 5%
• Small amount of reagents and samples 1-2 μL
• Various applications for electrochemical
determination
� Especially suitable for biosensors

Gold-Linked Electrochemical Immuno-Assay
(GLEIA, PCT/JP2007/56992)
Gold nanoparticle redox signal enhancement for detection of antigens
After the recognition reaction between the surface-immobilized primary antibody and hCG, the captured antigen
was sandwiched with a secondary antibody that was labeled with gold-nanoparticles. Then the amount of
gold nanoparticles was determined by its redox signal.
The primary antibody was Immobilized
directly on the working electrode, and
a sandwich-type immuno-reaction was
performed
A high potential was applied
In HCl for the oxidation of
gold nanoparticles
The voltammetric measurement
was performed.
DPV* : differential pulse voltanmetry
Reference:
• Direct fabrication of catalytic metal nanoparticles onto the surface of a screen-printed carbon electrode, Electrochemistry Comunications 8, p1375-
1380 (2006).
• Gold nanoparticle-based redox signal enhancement for sensitive detection of human chorionic gonadotropin hormone, Koutarou Idegami, Miyuki
Chikae, Kagan Kerman, Naoki Nagatani, Teruko Yuhi, Taturo Endo, and Eiichi Tamiya, Electroanalysis 20, p14-21 (2008)

Current (μA)
Application of
Gold-Linked Electrochemical Immuno-Assay I
Typical differential pulse
voltammograms of GLEIA.
Potential (V)
1000pg/m
500pg/ml
250pg/ml
125pg/ml
62.5pg/ml
0pg/ml
Human Chorionic Gonadotropin Hormone standard curves
constructed with buffer (●) and with serum (●)
Current (μA)
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Sample vlume:1 – 1.5μL
Lower detection limit: 62.5 pg/mL
0 62.5 100 1000
hCG concn. (pg/mL)
control1 R 2 =0.9996
serum R 2 =0.9971

Current (µA)
Application of
Gold-Linked Electrochemical Immuno-Assay II
1.2
1.0
0.8
0.6
0.4
Insulin standard curve constructed with human serum
R^2 = 0.99713
0 100 300 1000
Insulin (pg/mL)
Sample vlume:1.4μL
Lower detection limit: 125 pg/mL

DNA sensor
Gene aggregation method(patent No.364257
4)
A B
100nm 100nm
(Low concentration of DNA
or Before amplification)
(High concentration of DNA
or After amplification)

Application of DNA-sensor I by LSV
invA gene of salmonella bacterium
(PCR)
M a b
2.5
1.5
0.5
1
0.8
0.6
0.4
0.2
O157 gene (LAMP)
M a b c d e
2
1
negative
0
100 200 300 400 500 600 700
negative
positive
positive
0
100 200 300 400 500 600 700
a
b
c
d
e
Genetically-modified corn (LAMP)
M a b c
2.5
2
1.5
1
0.5
M a b c
0
200 300 400 500 600 700 800
2
1.6
1.2
0.8
0.4
negative
Hepatitis B virus gene from blood
sample (Nested PCR)
negative
positive
positive
0
200 300 400 500 600 700 800
a
b
c
a
b
c

Application of DNA-sensor II
Many people have tried to develop techniques to detect and identify species
origin especially in food products during the last decades which are very
important due to the Economic, Health, and Religious issues.
Pork contents as a food
Raw meat Raw sausage Raw lard Coocked
sausages

Application of DNA-sensor II
Electrochemical genosensor for the detection of pork’s 12S rRNA gene
using chronoamperometry (CA)

Conclusion
� The sensitivity of our immunochromatographic tests is at least
several times higher than those of conventional tests.
� DEP-Chips are suitable for POCT because of their ease to use,
low cost, disposability, economy of reagents and samples.
� GLEIA with DEP-Chips is a new sensitive immunoassay and will
provide a new POCT.
� Genosensors with DEP-Chips can determine the existence of
target genes within 5 min after amplification by PCR or LAMP.
The genosensor will provide a new POCT for safe of foods and
environments etc.
Contact information: Hiromi Ushijima,
TEL/FAX +81-761-51-7210
postmaster@biodevicetech.com