An Integrated Capacitive Array Biosensor for the Selective and

Real-Time Detection of Whole Bacterial Cells

Numa Couniot, Laurent A. Francis, D. Flandre

laurent.francis@uclouvain.be

4th International Symposium on Sensor Science July 13-15, 2015, Basel

Problem definition and Challenges

Matrix Detec+on  levels In  27  nL

Blood 1  bacteria/mL 1  bacterium  (p  =  0.003%)

Breast  milk 103  bacteria/mL 1  bacterium  (p  =  3%)

Urine  samples 105  bacteria/mL 3  bacteria

•  How can we detect such a little amount of bacteria?

For a 300 µm x 300 µm sensor in a 300 µm-thick channel

•  Selectivity?

•  How to deal with different solutions?

1 Staphylococcus aureus/mL must be detected among:

•  109 red blood cells/mL •  Possibly other non-pathogen bacteria

Known problem for electronic biosensors (FET, impedance, etc.): à the screening from the surface properties (e.g. electrical double layer) at high salinity

Problem definition and Challenges

Electrode Double Layer (DL)

~ 1-30 nm

Bacterial cell ~ 1 µm Electrolyte

•  How to deal with different type of solutions/electrolytes?

Electrical potential

- - - - - - - - - + + + + + + + + + + + + + - - - - - - - -

- - - + + +

Strongly depend on the ionic strength!!!

Problem definition and Challenges

Electrode Double Layer (DL)

~ 1-30 nm

Bacterial cell ~ 1 µm Electrolyte

Surface effects à Low sensitivity

Volume effects à High sensitivity

Go  to   High  Frequency!

1  bact.  =  ~70  aF

•  How to deal with different type of solutions/electrolytes?

Strongly depend on the ionic strength!!!

1 µm

Bacteria

Transducer

Selective agent

Readout interface

Staphylococcus epidermidis

à Similar to S. aureus à Non-pathogenic

Interdigitated microelectrodes à High active area à Similar size as bacteria à Electric field in surface

Lysostaphin à  Selectively destroys

bacterial cell wall à  Extendable to most

bacteria with lysins

CMOS à  Low-cost à  Miniaturization à  System integration

BIOLOGY

SENSOR

BIO/CHEM.

ELECTRONICS

CMOS capacitive array biosensor

Our approach

Electrolyte capacitance monitoring

Interdigitated microelectrodes

(IDE)

Integrated pixels

Integrated oscillator

Electrokinetic effects

Four steps to get to bacterium detection

1

2 3

4

Interdigitated microelectrodes

Interdigitated microelectrodes (IDE)

[Couniot et al., Biosensors and Bioelectronics, 67, pp. 154-161, 2015]

TOP  VIEW CROSS  SECTION

+50 mV

-50 mV

+I0

-I0

+50 mV

-50 mV

Cins Rsol Cins

Csol

ALD-alumina passivated electrodes

Time [min]

Y/ω

[pF]

# Ba

cter

ia [#

/mm

2 ] Optical

Electrical

[Couniot et al., Biosensors and Bioelectronics, 67, pp. 154-161, 2015]

Real-time monitoring of S. epidermidis

No background noise Low-cost Robust to wash

Advantages:

Selectivity based on lytic enzymes (lysostaphin)

[Couniot et al., Biosensors and Bioelectronics, 67, pp. 154-161, 2015]

Destroyed S. epidermidis causes decrease in capacitance

Selectivity means based on lytic enzymes Urine with S. epidermidis (target) and E. faecium (control -)

[Couniot et al., Biosensors and Bioelectronics, 67, pp. 154-161, 2015]

1.  Naked 2.  Ef 3.  Ef  +  Se 4.  Ef  +  Se(killed)

Same number of E. faecium, no impedance shift

[Couniot et al., Biosensors and Bioelectronics, 67, pp. 154-161, 2015]

Selectivity means based on lytic enzymes Urine with E. faecium only (control -)

1.  Naked 2.  Ef 3.  Ef(not  killed)

Selectivity means based on lytic enzymes Reproducibility of the normalized shift

Integrating electrokinetic effects

~ 1% of bacteria captured

~ 99% of bacteria lost

~ 1% of bacteria captured

~ 99% of bacteria captured

Trap bacterial cells with electrokinetics

How to decrease the LoD?

FLOW

FLOW

Macroelectrode For electrokinetic actuation

Capacitive Sensor

+7 V

- 7 V

50 mV

à  Generate electrokinetics (EK) effects

Design of the device

[Couniot et al., Lab on chip, submitted, 2015]

LoD : 3.5.105 CFU/mL in 20 min à 11x better than without EK

AC-Electroosmosis @ 10 kHz

[Couniot et al., Lab on chip, in Press, 2015]

Bacterial incubation

0 20 40 60 803.15

3.2

3 .25

3.3

3 .35

T im e [m in ]||Y

/ω||

[pF] w/ AC-EO

w/o AC-EO

7.10 CFU/mL6

1.6 . 10 CFU/mL7

~ 5

fF/m

in

~ 1 fF/min

PBS 1:1000 PBS 1:1000

LoD : 105 CFU/mL in 20 min à 38x better than without EK

OFF/ON Steps

0 20 40 60 803.15

3.2

3 .25

3.3

3 .35

3.4

3 .45

3.5

3 .55

T im e [m in ]||Y

/ω||

[pF]

w/ DEP & ET

w/o DEP & ET

Bacterial incubationPBS 1:1000 PBS 1:1000

7.10 CFU/mL6

1.6 . 10 CFU/mL7

Δ1

Δ2

Δ3

Δ4

Δ5

[Couniot et al., Lab on chip, in Press, 2015]

Electrothermal + Dielectrophoresis @ 63 MHz

à Flow-based method to direct bacteria from edge to the sensor center

VHF Capacitance-to-Frequency converter

VHF Capacitance-to-Frequency converter

[Couniot et al., IEEE TCAS II, vol. 62, 2, 2015]

Cins

CDL

Cins

CDL

Zbact

Csol

Rsol

Cpar

M5VHF GND

M4

M3

M2

M1 SiO2

CMOS

Al2O3DL

Electrolyte

VIA45

VIA

Si

Bacteria

VHF GND

CplCcyt

RcytCwall

CROSS  SECTION

Sensing Part

Electronics

VHF Capacitance-to-Frequency converter

[Couniot et al., IEEE TCAS II, vol. 62, 2, 2015]

TOP  VIEW

VHF Capacitance-to-Frequency converter

en

Vdd

Vdd Vdd Vdd

CL=10 pF

Five-stage ring oscillator Ten-stage frequency divider

Sub-interdigitated microelectrode arrays (M5)

Vout

fIDE

Five-stage ring oscillator

5 sub-interdigitated electrodes (M5)

Medium capacitance

[Couniot et al., IEEE TCAS II, vol. 62, 2, 2015]

Frequency divider

~  300  MHz

÷  1024

~  300  kHz

OUTPUT

0 0 0 1 1 1 0 1 0 1

Intrinsic and extrinsic capacitances

A factor 2 of difference despite the same coverage…

f200 µm−1 ∝ 1pF + 0.77*Csol,200 µm( )f100 µm−1 ∝ 0.8pF + 0.77*Csol,100 µm( )

3.5 ± 0.1 pF

1 ± 0.025 pF

The capacitance decreases à  Assessed by simulations/models

since cytoplasm dominates @ VHF à  εr,cyto ≈ 70 < εr,PBS = 80

Before After

Bacterial sensing in pure PBS

1060

Sens

itivi

ty [%

]

200 μm-sided: exp. #1exp. #2mean

100 μm-sided: exp. #1exp. #2mean

4

8

12

107 108 109

fIDE [Hz]~ 150 MHz

[Couniot et al., IEEE TCAS II, vol. 62, 2, 2015]

Capacitive biosensor array

Single bacterial cell

Reduce the sensor size

Bacterial binding

Δ1 Nominal sensor capacitance: 100%

Single bacterial cell

Δ2

[Couniot et al., IEEE TBCAS, in Press, 2015]

How to improve sensitivity & multiplexing?

Single bacterial cell

Problem: the bacterial cell can

be outside the sensor

Bacterial binding

Δ1 Nominal sensor capacitance: 100%

How to improve sensitivity & multiplexing?

Single bacterial cell

Δ2=0

[Couniot et al., IEEE TBCAS, in Press, 2015]

Single bacterial cell

Solution: make a sensor array

Bacterial binding

Δ1 Nominal sensor capacitance: 100%

Single bacterial cell

Δ2

[Couniot et al., IEEE TBCAS, in Press, 2015]

How to improve sensitivity & multiplexing?

System architecture (Top view)

[Couniot et al., IEEE TBCAS, in Press, 2015]

System architecture (correlated double sampling)

[Couniot et al., IEEE TBCAS, in Press, 2015]

Vdd

Vdd

VddMR

MBUF

MSELCIDE

CD

MC1 MC2

MINIT

Vc1 Vc2

MT

MSHR

Vshr

Vdd

MBUFR

MSELR

MBR

Vselc

CSHR

CLR

MSHS

Vshs

Vdd

MBUFS

MSELS

MBSVselc

CSHS

CLSMB

Vbr

Vbs

Vb

Vsel

Vr

Voutr

Vouts

Pixel(i,j)

ColumnAmplifier(j)

Outputstage

Columnbu

s

Vinit

VIDEVref

Vpix

Vinit

Vinit

VgT

Buffer + Row select.

Charge sharing principle

Subthresh. Gain

Csol

Cins

σsol

Ideal linearity

−50 0 500.12

0.16

0.2

0 .24

0.28

Variation of parameters [%]

VgT

[V]

σsol = 1.8 mS/mCsol = 55.6 fFCins = 500 fFRsol = 7 MΩCDL = 4.5 pF

Csol Rsol

Cins

CDL

CinsCDL

CIDE PIXEL TOP  VIEW

0 10 20 30 40 50−10

−5

0

5

Time [min]

Vou

t[mV

]

13

4

PBS 1:1000w/ bacteria

PBS 1:1000w/o bacteria

2

Pixel (2,7)

Pixel (2,6)

Pixel (2,8)

w/o bacteria w/ bacteria

Real-time monitoring

[Couniot et al., IEEE TBCAS, in Press, 2015]

w/o bacteria

w/ bacteria

Type 1

Type 2

w/o bacteria

w/ bacteria

Type 1

Type 2

*

w/o bacteria w/ bacteria

[Couniot et al., IEEE TBCAS, in Press, 2015]

Real-time monitoring

*

[Couniot et al., IEEE TBCAS, in Press, 2015]

Real-time monitoring

0Number of bacteria

Simulation

2 4 6 8 10 120

10

20

30

0

0.23

0.46

0.69

ΔVo

ut [m

V]

ΔC

sol [

fF]

Number of bacteria0

-20

Experimental

#5

ΔVo

ut [m

V]

5 10 15 20

0

20

40

#6

#7

#11#11

#23

#13

#20

#12#9

#10

#3

#8#8

VIDE

Vgnd

VIDE

Vgnd

≠

ΔC  ≈  167  aF ΔC  ≈  38  aF [Couniot et al., Sensors and Actuators B, vol. 189, pp. 43-51, 2013]

VIDE

Vgnd

≠

ΔC  ≈  7  aF

CMOS capacitive array biosensor

Interdigitated microelectrodes

(IDE)

Integrated pixels

Integrated oscillator

Electrokinetic effects

1

2 3

4

High sensitivity @ high salinity

High sensitivity By bacterial trapping Single Bacteria

Detection

Selectivity

F.R.S.-­‐FNRS  Funds D.  Bol,  J.  Mahillon  &  J-­‐L.  Gala  for  their  supervision

T.  Vanzieleghem  &  J.  Mahillon  for  their  biological  exper+se J.  Rasson  &  N.  Van-­‐Overstraeten  benefits  for  useful  discussions

O.  Poncelet  for  ALD  deposi+on C.A.  Dutu  for  technical  help  with  PDMS  cap  micro-­‐fabrica+on

D.  Spôte  for  the  fabrica+on  of  the  pressure  tool UCL  WINFAB  plaoorm  for  help  with  micro-­‐fabrica+on

UCL  WELCOME  plaoorm  for  help  with  measurement  setup

Thank you for your attention! Questions?

laurent.francis@uclouvain.be

Acknowledgements