A. Bongrain1, H. Uetsuka3, G. Lissorgues2, E. Scorsone1, L. Rousseau2, L. Valbin2, S. Saada1, C. Gesset1, P. Bergonzo1

1CEA, LIST, Laboratoire Capteur Diamant, GIF-SUR-YVETTE, F-91191, France.2 ESIEE – ESYCOM Université Paris Est, Cité Descartes, BP99, 93162 Noisy Le Grand, France.

3Diamond Research Center, AIST, Tsukuba, 305-8568, Japan

Measurement of DNA hybridization based on diamond micro-cantilever sensing

I. Introduction

MEMS structures can offer a real improvement for bio-detection applications in term of cost, miniaturization and sensitivity

Diamond exhibits advantageous properties for MEMS-based bio-sensing applications

-mechanical properties (High Young modulus, High resistance to fracture)

-chemical properties (bio-chemical inert, covalent bonding using carbon chemistry)

II. Diamond MEMS fabrication process IV. Cantilever’s resonance frequency measuring set up

III. DNA grafting process on boron doped diamond cantilevers

V. Results and discussion

Detector anddemodulator

Piezo-electric cell driven voltage(frequency scan)

Readout signal

Spectrum analyser

Laser beam

Piezo-electric cell

DNA attachment on boron doped diamond cantilevers

Conclusion and perspectives

Optimization of diamond nano-particules plasma etching duration optimization (step 4)

Diamond nano-particules etching duration (min)

Nan

o di

amon

d re

sidu

al d

ensi

ty (

cm-2)

10 µm

10 µm

10 µm

O2/Ar

Metal hard mask deposition on an oxidized Si substrate

Moulds etching by DRIE*and metal removing

Diamond nano-particules spreading

Metal hard mask depositioninside the moulds and diamond

nano-particules etching

Metal hard mask removingand Diamond growth

(MPCVD)

Metal tracks deposition Structures releasingBack side DRIE

Gas injection (H2, CH4)

Gas extraction

Microwave guide

Microwave plasma

Substrate support

Polycrystalline diamond synthesized by MPCVD (Microwave Plasma Chemical Vapor Deposition)

Ionization of a H2/CH4 gas mixture (99:1) by microwave

CH4 supplies carbon atomsH2 prevent from graphitic carbon growth

MPCVD growth reactor for diamond growth on large surface (until 4 inches)

SiO2 chemical etchingand short BDD* growth

100 µm

10 µm10 µm

4 inches

1) 2) 3) 4)

5) 6) 7) 8)

- Versatile process

- Adapted for largesurface

- No diamond etching or polishing required

*BDD: Boron Doped Diamond

*DRIE: Deep Reactive Ion Etching

Cyclic voltammetry

Chemical reaction

Hydrogen or oxygenterminated surface ofboron doped diamond

Diazonium salt electrochemicalreduction

Electrochemical reduction of –NO2

Attachment of cross-linker andthiol modified ss-DNA grafting

Hybridization withcomplementary targetDNA functionalized with a color maker

Cyclic voltammetry

Fluorescent tag

Cyclic voltametry of NO2 to NH2 reductionin KCL 0.1M in DI water:ethanol (9:1)

First cycle

Four-step electrochemical DNA grafting process

Attachment of 32-base DNA

95

96

97

98

99

100

101

1 2Before denaturation After denaturation

Re

fere

nce

Re

fere

nce

Me

asu

rin

g

Me

asu

rin

g

97.45%

99.74%100% 100%

Rel

ativ

e va

riatio

n of

the

reso

nanc

e fr

eque

ncy

(%)

2.3 %

7 mm

Liquid cell

- Resonance frequency measured by Doppler laser interferometry

- Cantilevers actuated by an external piezo-electric cell

- Measurements in liquid

2 identical cantilevers on the chip

1 reference (bare cantilever)

1 measuring (functionalized)

a- DNA grafting success checked by fluorescence after hybridization

Fluorescence represented by the green light indicates that attached DNA is located on the measuring cantilever, only.

Significant contrast between grafted area and non grafted area.

b- Comparison of cantilever’s resonance frequency between before and after denaturation

1. First measurements in a Phosphate Buffer Solution (PBS) volume of 400 µL on both reference and measuring cantilevers, respectively.

2. Denaturation in NaOH

3. Second measurements in a purged PBS volume of 400 µL on both reference and measuring cantilevers, respectively.

Significant decrease of the resonance frequency measured on the measuring cantilever after denaturation

Same resonance frequency measured on the reference cantilever after denaturation

Differential measurement of -75 Hz (2.3% of a 3 kHz-cantilever)

c- Discussion

Denaturation

Decrease of electrostatic repulsions

Hybridization

Diamond and Biology C.E. Nebel et al, (2007).

Measured resonance frequency shift mainly attributed to the surface stress contribution because:

-The resonance frequency shift is negative and hence opposed that it should be if mass were the dominant contribution

-Cantilever itself as a poor sensitivity to a mass change in liquid (cantilever length: 900 µm)

-Calculations showed that the grafted DNA layer as an unsignificanteffect on the overall cantilever Young modulus

-DNA is negativelly charged (sugar phosphate) and hence electrostaticrepulsions are induced between grated DNA strand

-High cantilever sensitivity over surface stress change (~tens Hz/N/m)

Detection of DNA denaturation by measuring cantilever’s resonance frequency shift

Resonance frequency shift mainly attributed to a change of surface stress induced by electrostatic repulsons between grafted DNA

Hybridization cycleFlu

ores

cenc

e in

tens

ity (

arbi

trar

y un

its)

Yang et al.., Nature Materials, 1 (2002) 253

High structures sensitivity in dynamic mode

Robust devices

Stability of surface functionalization

Transducers resistant to harsh chemical environment

Cyclic voltametry of diazonium reduction on boron doped diamond

Resonance frequency shift mainly attributed to a change of surface stress induced by electrostatic repulsons between grafted DNA

Cantilevers sensitivity over surface stress (~tens Hz/N/m) offers large opportunities of bio-sensing applications (typical induced surface stress change ~tens mN/m)