Biotronics 20101028

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Highly sensitivenucleic acid based bacteria detection

using single chamber system

Junhong Min

Laboratory ofMedical andEnvironmentalAnalysis System

College of Bionano Technology, Kyungwon University

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Contents

Introduction

DNA concentration + amplification + detection

RNA concentration + amplification + detection

Bacteria concentration + lysis + amplification + detection

Conclusions

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BIOTronics 2010

Motivation I - Complexity

Nucleic acid (NA) based bacteria detection requires sophisticated

serial processes

Stanford bioengineering

Professor Stephen Quake.

Wonderful microchips

Simple chipI can fabricate

Sample preparation Amplification

Detection

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CellConcentration

Celllysis

NApurification

NAamplification Detection
http://news-service.stanford.edu/news/2006/january18/gifs/fluidicschip.jpg

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Motivation II Volume and Sensitivity

Environmental sample has too large volume to be introduced into

micochip format sensor.

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1LSample volume 100mL ~ 100L

Sample volume needs to be reduced with sustaining an amount of

analyte in order to achieve high sensitivity

SampleLimit

(MPN)

Sample

vol.River (I) < 50 100mL

Sea Area (I)

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Purpose

To develop simple single chamber system

including functions of

1. Concentration (Nucleic acid or Bacteria) or Isolation

2. Lysis ( for bacteria)

3. Analyte amplification

4. Signal generation

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Target samples

E. coli is often preferred as an indicator organism because it is

specific for and reliably reflects fecal contamination

Particularly, E.coli O157:H7is one of representing pathogenic

bacteria.

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Design of Micro Chip

Conventional fabrication tech. Simple use

Considerations1. Micro PCR compatible

2. Chip based

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- 0.5 l sample reguired- Detection time : 30 min

- Si-Water based chip

- SyBr green based real time detection

Micro-PCR

Biosensors and bioelectronics, 21, 2161-2169, 2006

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Design of Lab In Tube(LIT)

100m

Fast sample loading: 2 mL/min No bead loss No chip damage

1) Sample loading component

2) Fluorescence signalmeasurement component

3) RNA or Bacteria adsorption andNASBA reaction component

Considerations1. Pressure drops

2. Friendly design (E-tube type)

1)

2)

3)

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Pressure drop 9/36

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Introduction



Bacteria concentration + lysis+amplification + detection

Conclusions

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Concepts_ E.coli detection with DNA

1. How to adsorb DNA on micro pillars in micro chip

2. How to amplify DNA adsorbed with microstructure (large surface area)

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Sample loading

NA concentrationSample (1mL)

NA amplification

Bacteria lysis

Fluorescence read out

Single chamber

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DNA adsorption 12/36

DNA concentration

DNA amplification usingmicro PCR


Single chamber- Nucleic acid could be fully hydrated in

presence of kosmotropic salt

- Nucleic acid can be released from silica

surface at high pH.

- Simple

- No toxic salts such as chaotropic salt

- No special surface

Indirect binding of Nucleic acid

Hydrophilic interaction

With

kosmotropic salt

Without

kosmotropic salt
http://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=G

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DNA Adsorption on silica surface 13/36

Kosmotropic salt effect Surface effect

l f ll h

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Amplification in micro pillar chip 14/36

DNA d i d lifi i i hi

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DNA adsorption and amplification in a chip 15/36

Chip optimization

S l l di t l

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Sample loading tools 16/36

DNA t ti ff t i i l hi

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DNA concentration effect in single chip 17/36

Lab chip, 2010, accepted

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Introduction



Bacteria concentration + lysis+amplification + detection

Conclusions

RNA adsorption on microbead in a tube

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RNA adsorption on microbead in a tube

NA concentration

NA amplificationby NASBA


Single chamber - Silica has very low pKa (pH=2).- Nucleic acid can be absorbed on silica

surface at low pH.

- Nucleic acid can be released from silica

surface at high pH.

- Simple

- No special salt

- No special surface

Simple binding of Nucleic acid

Modified Charge Switch Tech.

At low pH At high pH

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RNA Adsorption on 100um beads in tube
http://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=G

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RNA Adsorption on 100um beads in tube

0

20

40

60

80

100

3 5 7 9

RNA

adsorption

rate

[%]

Buffer solution [pH]

250 500

1000 5000

The number of beads

Amplicon

concentration[ng/l] 50

40

30

20

10

0

pH 3 pH 5

-100-200-300 100 200 3000

1000

2000

3000

4000

FluorescenceIntensity(A

.U.)

x (m)

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NASBA in conical tube with micro beads

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NASBA in conical tube with micro beads

200 bases

200 bases

200 bases

Beads number

BSA

concentration

Beads number

with BSA 0.3%

PC 250 500 1000 5000 NC

PC 0.5% 0.3% 0.1% 0.05% 0% NC

PC S 250 500 1000 5000 NC

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Sensitivity test

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Sensitivity test

L 1 2 3 4 5 6 7 8 910 11 12

Before After

PC 102 101 102 101 100 NC

Sample : 100mL of rain water

102CFU/100mL 100 CFU/100mL

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w/o concentration With concentration

Direct detection of E coli O157:H7

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Direct detection ofE.coli O157:H7

0

10000

20000

30000

NC 0 1 2

Fluorescen

ce

Intensity

[A.

U.]

Cell Concentration [c.f.u/100ml]

Rain-100nMRain-200nM

River-200nM

*

NC 100 101 102

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Biosensors and bioelectronics, 26, 112-117, 2010

Gathering environmental

sample at Han Liver

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Introduction



Bacteria concentration + lysis + amplification + detection

Conclusions

Bacteria concentration

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RNA amplificationby NASBA with molecular

probe


Single chamber

Bacteria lysis

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- Simple binding of bacteria

- IF at low pH, Bacteria tends to be

aggregated on hydrophilic surfaces.

- Some salt can help bacteria fusion.

Self aggregations by hydrationsLysis by micro Inductive heating

At low pH

Metal

Hydrophilic surface

Bacteria

Sample

Bacteria lysis Induction heating

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Bacteria lysis _ Induction heating


RNA amplificationby NASBA with molecular

probe


Single chamber

Bacteria lysis

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- No need to perfect contact

- Local heating available

- Multifunctional micro-beads

- Local heat generation

- No RNA degradations

Micro Induction Heatings

micro heat induction

Sample

Metal

Hydrophobicity on bacteria adsorption 27/36
http://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=Ghttp://images.google.co.kr/imgres?imgurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.gif&imgrefurl=http://www.csb.yale.edu/userguides/graphics/ribbons/help/dna_rgb.html&h=488&w=610&sz=36&hl=ko&start=3&tbnid=MQIFLXuLMYJ_JM:&tbnh=109&tbnw=136&prev=/images?q=DNA&gbv=2&hl=ko&newwindow=1&sa=G

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Hydrophobicity on bacteria adsorption

0

20

40

60

80

100

TEOS APTS TEB TDF HDF

E.coliadso

rption

rate[%]

5 30 60 85 110

1m

Contact

angle

0

20

40

60

80

100

20 50 100 200 500 1000

E.coliadsorption

rate[%]

The number of beads

TEOS

105 c.f.u/10 mL ofE.coli

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Low concentration E.coli adsorption

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Low concentration E.coli adsorption

0

20

40

60

80

100

102cfu 101cfu 100cfu

Bacteria

adsorp

tion

rate

[%]

Bacteria concentration [c.f.u]

E.coli

Bacillus

102 101 100

O157:H7

B.cereus

105 cfu E.coli

concentration rate

In case of 102 c.f.u/10mL,

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Aquatic chemistry optimization 29/36

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Aquatic chemistry optimization

0

20

40

60

80

100

0 10 100 0 10 100

Bacteria

ads

orption

rate

[%]

MgCl2 [mM] Na2SO4 [mM]

0% PEG

1% PEG

E.coli O157:H7 B.cereus

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Cell lysis on micro bead 30/36

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Cell lysis on micro bead 30/36

NikelIron

Copper

Lab chip, 10, 909~917, 2010

Measuring device 31/36

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Measuring device 31/36

Pump

Micro induction heating device

Optical device

- 30 X 30 X 20 cm3

- Battery operated

- Temp. and flow rate controllable- LCD Display

E.Coli Measurement 32/36

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Before After

PC 102 102 101 100 NC101

E. coli in the concentration of 101 c.f.u/100 ml could be detected by bacteria

concentration using the single plastic tube chamber

Chop &stomach

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In Situ Detection ofE.coli Using Beacon

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g

NC 0 1 2

Fluoresce

nce

intensity

[A.U

.]

E.coli concentration [c.f.u/100ml]

100 101 102

Signal : 22795 A.U.

S&R : 50.88

The proposed method (bacteria concentration, cell lysis, mRNA amplification and signaling in single

chamber) could successfully detect less than 10 E.coli O157:H7 in 1 g of beef (averaged SNR = 7.7).

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Biosensors and bioelectronics, 2010, In press

Conclusion 34/36

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Sample concentration, cell lysis, amplification, and detection

processes could be performed with single chamber containingmicro beads or micro pillar chips.

Low concentration (~100

c.f.u/100ml) of pathogenic bacteria (E.coli O157:H7)in large volume could be detected with single

polymer chamber within 3 hours.

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Acknowledgment 35/36

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g

Korea Environmental Industry and Technology Institute Ministry of Environment as

The Eco-Technopia 21 project

Korea Research Foundation Grant funded by the Korea Government (MOEHRD)

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Students

Ji-Yeong WonSoyeon KimBinh TranSohye Han

ResearchersJa Yeon KimSeung Hee Chung

Collaborators

Prof. Jung-Hwan Park (Kyungwon U.)Prof. Sang Jun Son (Kyungwon U.)Prof. Young Rok Kim (Kyunghee U.)

Funds

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Thanks for your attention

Documents

Biotronics 20101028