seminar 2 copy

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A personalizedFOOD ALLERGEN TESTING PLATFORM on a cellphone

Presenter:

NGUYEN VAN HAU

Ahmet F. Coskun et al. Lap chip, 2013

INTRODUCTION

Food allergen overviewDetecting peanut allergen

by ELISA methodSmartphone-based colorimeter

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Allergy : a hypersensitivity disorder of the human immune system to harmless substance

Allergen : protein that causes allergenic reaction

Allergy, allergen, common symptomsFOOD ALLERGY

Skin reaction Digestive problem Fightening of chest Stuffy nose Dizziness Itchiness and swelling of the

mouth throat

Common symptoms

Source: National Institute of Allergy and Infectious Diseases (NIAID); Food allergy research & education (FARE)

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Each year, around 150 to 200 people die in the US, around 10 people in the UK die because of food allergies Yearly, allergies cost an estimated $18 Billion to the US

4% adults(6 million)EFFECT OF FOOD

ALLERGY TO WORLD

POPULATION

8% children

(9 million)

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FOOD ALLERGY

1. Milk and egg : 0.2-0.4%*

2. Crustacean shellfish : 1.2%

3. Fish : 0.4%4. Peanut : 0.6-

1.3%5. Tree nuts : 0.4-

0.6%6. Soy7. Wheat

* among US population

Big8 allergenic foods (90% of food allergy)

Source: National Institute of Allergy and Infectious Diseases (NIAID); Food allergy research & education (FARE)

0.6% of world population has peanut allergy Less common in Asia and Africa In US, 1 in 90 people and 4 in 100 children has

peanut allergy > 15% of children get food allergen in the

school

Peanut allergy

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13 proteins in peanut can cause allergy from Ara h1 to Ara h13 (Ara h: Arachis Hypogaea)

Ara h1 and Ara h2 is the main allergenic proteins (Ara h1: 12-16%; Ara h2: 5.9-9.3% total peanut proteins content)

Bublin & Breiteneder, 2014; Saiz et al., 2013; Scurlock & Burks, 2004; Koppelman et al., 2001.

PEANUTS ALLERGY

Peanut containing foods Peanut butter Baked goods (e.g.

cakes, muffins) Cake icing Candy Cereals Ice cream chocolate

Allergenic proteins in peanut

Hidden concentr

ation

Impurity of raw material

Contamination in manufacturing

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ALLERGEN DETECTION METHODS

Unsuitable for personal use in publish settings

Polymerase chain reaction (PCR)

Mass spectroscopy (MS) Antibody immunoassays Surface-plasmon-resonance

(SPR) Biosensor Array immunoassay Electrochemical

immunosensors

Allergen = Protein

7http://www.elisa-antibody.com/

Allergenic protein = antigen

Biotin

Substrate

Polystyrene surface

Detection antibodyCapture antibody

Enzyme

SANDWICH ELISA METHOD

Total peanut proteins Major allergenic proteins (such as

Ara h1 or Ara h2)

(ELISA=Enzyme Linked ImmunoSorbent Assay)Principle

2 type of allergenic proteins detections

ADVA

NTAG

ES Quantitative and qualitative

Fairly rapid Highly sensitive

and selective Simple procedure Low cost

Comparing to other techniques, ELISA has some advantages

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UV-Vis spectrophotometer Bulk instrument

High cost Short

measurement time

High precision and accuracy

Portable instrument Low cost Short measurement

time (1 second) Comparable precision

and accuracy

Smartphone-based colorimeter

SMARTPHONE-BASED COLORIMETER

Source: http://www.colorimetrix.com/

Advantages

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UV-Vis spectrophotometer Smartphone camera

A=−log (IIo )= εbcBeer-Lambert law

Light beam

Grating

Absorption

spectrum

Light beam

Photodiode array detector Camera

sensor

SMARTPHONE-BASED COLORIMETERFundamental

Increase [peanut]

The color intensity of the image depend on the intensity of light that the detector recieve

Absorbance

Color intensity

[peanut] absorbance

[peanut] color intensity

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Design a smartphone-based colorimeter

for detecting and qualifying peanut allergen

OBJECTIVE

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EXPERIMENTAL

Peanut allergen ELISA test kitProposed smartphone-based colorimeter

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EXPERIMENTAL

Overview about the experiment

Analyte : total peanut proteins Sample : cookies Standard : peanut proteins Colorimetric method : commercial ELISA peanut allergen test kit

(such as Veratox, Neogen, 8430) Detection method : proposed smartphone-based colorimeter

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A peanut allergen test kit (such as Veratox, Neogen, 8430) consists of: 48 mixing wells 48 antibodies coated wells 5 standard solutions (1, 2.5, 5, 10, 25 ppm) Conjugate Substrate Stop solution

PEANUT ALLERGEN COLORIMETRIC DETECTION

ELISA test kit

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VERATOX peanut allergen test kit information Standard prepare from : 22 varieties of raw

and roasted peanuts Antibody detected : Peanut proteins Results reported as : Total peanut

proteins concentration Range of quantification : 2.5-25 ppm (100-

1000 ng/mL) Extraction : 1 to 25 in 10mM PBS

buffer 15 min in shaker water bath (600C)

PEANUT ALLERGEN ELISA TEST KIT

Test kit information

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1 2 3 4

Grind the sample

Extraction with hot water (50-60oC) + PBS bufferAdd 3 drops of supernatant into

antibodies coated tubeIncubate for 10 min wash with bufferAdd 3 drops of

conjugate

Add 3 drops of substrate

Measure

MESUREMENT PROCEDURE

Capture antibodiesAllergen (=antigen)

Detection antibodies (linked with an enzyme)Reading solution

Incubate for 10 min wash with buffer

Incubate for 10 min wash with bufferAdd 3 drops of stop solution

http://www.elisa-antibody.com/

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MAIN PLATFORMSample

tube

Reference tube

Red LED

Light diffuser

Plano-convex lens

Reference

Sample

DESIGN & DEVELOP SYSTEM

Conceptual design

= constants

[peanut]

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Conceptual design

Light source LED=assay =650 nm

sensitivity

Red LED

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Conceptual design

Make the light from LED more homogenous

LIGHT DIFFUSER

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Reduce interference

APERTURE


Conceptual design

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Polystyrene test tube

(8mm 12 mm)

SOLUTION CONTAINER


Conceptual design

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Reduce interference

APERTURE (1.5 mm diameter)


Conceptual design

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Fit reference and sample tube into camera field-of-view

PLANO-CONVEX LENS

DESIGN & DEVELOP INSTRUMENTConceptual design

Ref Sample

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Platform is designed by AUTODESK INVENTOR software

Platform is fabricated by 3D printer


Fabrication platform

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[peanut] < 1 ppm : negative value

[peanut] 1 ppm : positive value

Sample

Ref


Android based smart application

[peanut] 5ppm can cause peanut allergyM. Morisse et

al. 2003

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Ref Sample

Original image

Ref Sample

Grayscale image

Relative Absorbance

Peanut concentration

CALIBRATION CURVE of standard

Sensing area

Ref Sample

Digital processing of tube images

A=log10Meanref1.15×Meansample

RELATIVE ABSORBANCE

Meanref, Meansample: average color intensity value inside rectangular zone

1.15: Normalization factor


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RESULTS & DISCUSSION

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Measurement range : 1-25 ppm peanut

Calibration curve : A = 0.028*C

R2 = 0.99 LOD : 1ppm


Analytical performance

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1. Peanut butter chocolate (PBC)

The proposed smartphone-based colorimeter is suitable for peanut allergent detection


Applying system with cookies samples

12 ppm, positive value

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2. Oatmeal raisin with walnut (ORW)

The system is specific with peanuts allergen testing



< 1 ppm, negative value

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3. Milk chocolate chip (MCC)

The proposed smartphone-based colorimeter is suitable for peanut allergent detection

The system is specific with peanuts allergen testing



< 1 ppm, negative value

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CONCLUSION

ELISA SMARTPHONE

Advantages of proposed system:

Highly sensitive and selective

Enable qualitative and quantitative

Portable Low cost system Personalized allergen

testing tool Easy-to-use Rapid analysis time

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CONCLUSIONBenefits of proposed system:

Proposed a system which enable detect and quantify allergen concentration is food products based on smartphone camera

The proposed instrument is useful for public health in various settings, including e.g., schools, restaurants, airplanes

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Acknowledgements

Assoc. Prof. Dr. Napaporn Youngvises

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Thanks for your kind attention

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Your questions is welcome

seminar 2 copy

Engineering