Biosensors and High-Throughput

Analysis
Aurélie Bouchet-Spinelli

CEA Grenoble
Part 2- Transduction methods
Electrochemical biosensing
Biorecognition Electrode
Layer potentiostat
transducer

Redox process
Steric process Signal
Change in the local redox state of the medium, in
the mass transfer behaviour or in the charge Potential
Current
transfer behaviour
Frequency
Advantages Conductivity
Real time measurements
Parralelised detection
Miniaturisation, integration Direct method
Signal processing Electrodes or µelectrode
Compatibility with thin film techno. Ion sensitive electrode
In vivo measurements ChemFET
Material costs Microscopy (SECM)
Electrode array
Drawbacks
Reference electrode Label based methods
Electrical interferences Redox labels
Electrochemical interferences Steric hindrance
Biocatalytic label
Optical biosensing
Biorecognition
S Optical transducer Light detector
Layer

Optical Path
Signal
Change in the optical properties due to molecular
binding or reactions Absorption
Fluorescence
Advantages Refractive index
No reference electrode Bioluminescence
Absence of electrical interferences …
Real time measurements
Multi-wavelenght analysis Direct method
In vivo measurements Optical fibres
Well defined energy levels Internal reflection spectro
Parralelised detection SPR, ellipsometry

Drawbacks Label based methods

Double beam methodologies Optical fibres
Background lights Microscopy
Signal processing
Miniaturisation, integration
Bleaching process
Optical transduction : molecular absorption
UV-Visible Spectroscopy Optical pathway cm
A = .l.C Beer’s law
Concentration mol.l-1
Molecular absorbtion coefficient
l.mol-1.cm-1

IR Spectroscopy

Transducer : optical fibres (Optrodes)

Optical transduction: Fluorescence
UV-Visible Absorption generation of an excited state
Relaxation to the fundamental energetic state
Non radiative behaviour (Vibronic relaxation  T°)
Radiative behaviour Emission

Fluorescence Phosphorescence
Trnasient (ns) Slow (>s)
Analysis ?
I F  KI0C

Transduction (Otical fibre, Waveguide) or detection (microscopy)

Fluorescence microscopy
Sensivity
Spatial resolution Semi-quantitative
Positive signal Parasite fluorescence (substrate…)

Electrochemiluminescence
O
e- h HN NH
N-(4-
(4-aminobutyl)
aminobutyl)-N-ethylIso -luminol
C2H5 O

Au S (CH2)4-CO-NH-(CH2)4 -N
NH
biotin NH
7 x 10-2 cm2 Avidin
O
Pyr-ODN/Pyr =1/20000 ABEI-biotin
Tampon borate pH=10, 50 M H2O2 M. Billon et al, Bioelectrochem., in press

CCD
Camera
Cibles

Biological probe recognition detection

Fluorescence: the reference method
hn hn’

Biotinylated
DNA target Fluorophore

Semi-quantitative measurement
Fluorescence intensity (IF)
Given in grey level scale TŽmoin nŽgatif IF = 90 IF = 110
Reference test IF =10
Microscopie ˆ fluorescence, image capturŽe
par camŽra CCD
Amplification (Fluorescent labelled nanoparticles)

! Bleaching process
! Fluorescent behaviour of many
supports (resins, substrate…) : Background
Silica beads
! Target labelling in general (change in molecular recognition ?)
FRET based photoluminescence

Quenching
Highly fluorescent
Low background
Homogeneous phase
Applicable to µ-fluidics

Labelled target or not

Highly fluorescent

Poly (p-phenylenethynylene) chromophore

Quenching
Highly fluorescent

FRET : fluorescence resonant energy transfer

Kim et al, Adv. Funct. Mat. , 2007, 17, 2780
DNA engineering for conformational response

Probe optical labelling: molecular beacon = « DNA loop »

Single molecule
hybridization
imaging

Yao et al, Chemistry 9 2003 5686. Photobleaching !

Probe optical labelling: DNA nanoswitch

Homogeneous phase
Applicable to µ-fluidics
Low background (slow response)
No target labelling
Buck et al, Anal.Chem. 79 2007 4724
Electrochemiluminescence detection
h
2500 ECL plot 1

signal (nb coups/50ms)

2000

1500

1000

h 500

0
0 2 4 6 8 10 12
temps (s)

Specific Non specific

e-
500
ECL plot 9
450
400

signal (nbs coups/50ms)

350
300
250
200
150
100
50
0

E
0 2 4 6 8 10 12 14
tem ps (s)

Fluorescence

O
e- h HN NH
N-(4-
(4-aminobutyl)
aminobutyl)-N-ethylIso -luminol
C2H5 O

Au S (CH2)4-CO-NH-(CH2)4 -N
NH
biotin NH
7 x 10-2 cm2 Avidin
O
Pyr-ODN/Pyr =1/20000 ABEI-biotin M. Billon et al, Bioelectrochem 66 2005 139
Tampon borate pH=10, 50 M H2O2
Chemiluminescence detection

Low cost CMOS photodetector array as a

solid support for a DNA chip, coupled with
revelation by enzyme-catalysed
chemiluminescence

Marchand et al, Biosens. Bioelectro. 20 2005 1813

What’s the signal ?
Molecular recognition
(No metabolic activity)

e-? What has changed?

E, i, Z, Y, 
How to label the recognition event?

Signal Direct Indirect

End point DNA electroactivity Fluorescence

detection Impedance Redox intercalators Labelling
Conductivity Accumulation
Microparticles
Amplification
Enzymes
Real time QCM Matrix
Transduction SPR or
Photocurrent… Labell
Direct methods: How to read recognition?

Probe labelling
Optical
Net Charge
Redox
refractive index
permitivity
Intrinsec conductivity

Electroactivity
Guanosine
Adénosine
Wheighting Desoxyribose
Stericity of the double strand
Mechanics of the double strand
Optical transduction by surface plasmon resonance
Optical transduction by surface plasmon resonance
SPR gives access to:

- Kinetic and thermodynamic

constants
-Multiplexing
- Real-time monitoring
- No label
-Not portable

Detection limit: femtomoles/cm2

 constant
Optical transduction by surface plasmon resonance

Head : A. Buhot

> 100 spots / chip

Interactions with:

- Lectins
- Enzymes
- Bacteria
- …

Research thematics about SPRi

sensitivity improvements
DNA SPRi optical transduction

Sur chaque spot

0 .5

ReflectivitŽ
0.4

Non complementary
0.3

R
0.2

0.1
0 PBS
0.0

0 5 10 15 20
Complementary
Angle dÕIncidence (deg.)
PBS

ReflectivitŽ
0.12

0.10

At 0
0.08

0.06
R
0.04

0.02

0.00
0 5 10 15 20

Temps (min.)

Guedon et al, Anal Chem. 72 2000 6003

Polypyrrole, peptides and SPR imaging
Anti-virus antibodies of hepatitis C detection in human sera
Antibody maping as markers of illness evolution
Biochip containing 11 HCV peptides

Sample 1

Sample 2
Recognition of different families
C 1-21 of
C 20-40
18 antibodiesC 131-150 E1
E2 NS2
14 NS3 NS4/1
NS4/2 NS5/1
Reflectivity (%)

NS5/2 Ova
10
Cherif et al. Clin. Chem. 2006

-2 0 2764,768 6081,016 8788,928 11496,76 14223,768 18234,896

Time analysis
Continuous and label free multiparametric (s) in real time
First SPR/ chemistry compatible with real clinical samples
Technological transfer toward EFS (CHU Grenoble) via Genoptics
Optical transduction by surface plasmon resonance
Specific Interactions with lectins:

Key – Lock principle Access to :

- Specificity
+ = - Kinetics constants

Possible to mimic
multivalency

Interactions with bacteria:

No specificity but a
fingerprint for each
bacteria tested

Culture / Capture / Measure

Bulard et al. Anal. Chem.,
Bouguelia et al. Lab Chip, 2013,13, 4024 2015, 87 (3), 1804
Optical transduction by surface plasmon resonance
Other applications of sugars at CREAB:

Electronic tongues/noses: Cross-reactive interactions

Original strategy for a large set of receptors

Leading to auto-corrective and continuous signal

CXCL12α

Protein discrimination
Complex sample analysis

Hou et al. Angew. Chem., 2012, 124, 10540

Startup Aryballe created in 2014
Microgravimetric and µmechanical biosensing
Biorecognition Accousto-mechanical
Layer Resonator
transducer

Inertial mass
Surface energy Signal
Change in resonance frequency or in surface
energy due to molecular binding or reactions Frequency

Advantages
No reference electrode
Real time measurements
Parralelised detection Transduction modes
Miniaturisation, integration QCM
Signal processing SAW
Compatibility with thin film techno. …
(Vibrating) cantilever
Drawbacks Vibrating membrane
Vibrations sensitivity
Electrical interferences Labeling method
In vitro measurements only Wheighting labels
Visco-elastic coupling in solvents
Cantilever arrays for mechanical transduction

Deflection Dependancy in the

Operation in static mode media and in ionic
Cantilever deflection due to modification species
in surface energy

Cantilever lenght
Poisson constant of the material
M Guirardel, Thesis of the University of Toulouse, 2003 Difference of surface energy of both faces
Cantilever arrays for mechanical transduction
ODN 12 mŽres
Sensitivity 10-12 g

Fritz et al, Science 288 2000 316

Operation in static mode

Concentration dÕODN

Application protŽomique

Possibility to work in the

vibrating mode
Problem of damping in
solutions

deflection measured by
optical interferometry
Quartz Crystal Microbalance (BAW)
Quartz crystal microbalance (QCM)

AT-cut Quartz 2d
Resonance 

Different harmonic availables n
1,2,3…
Displacement in the thickness shear mode
Shear wave velocity Thickness d

nb nb N
f0  n n
 2d d

Frequency constant : 0.168 MHz cm for quartz

3200 m s-1 for quartz
f0  9 MHz for d= 0.2 mm

f d f M q 2 f 0 M q
d  and M q  q Sd so  
f0 d f0 Mq  q Sn b
Mass loading
1 M q 1 M q   2.64 g.cm 3
f  2 f 0   f0
2 2
q
d + d
 qn b S q N S
Sauerbrey Sensitivity 230 Hz µg-1 (f0= 10 MHz, S= 1 cm2)
Quartz Crystal Microbalance in liquids
Work in liquid
Viscosity of the medium
Accoustic coupling
Wave damping

1 reference surface in air, 2nd in the medium

Coupling affected by roughness, porosity,
rigidity of the coating
Rigid compact coatings Sauerbrey equation
Wet, rough or porous surfaces

Decay rate of the accoustic wave in Medium viscosity

liquid depends on its viscosity
3 ll
Damping phenomena = f attenuation f 0   f 0 2
Overestimation of mass loading  q  q
Quartz elasticity
q
Biological molecules nb  q
Viscoelesatic properties
Over-estimated m
Quartz Crystal Microbalance in liquids
QCM-D gives access to :
-Mechanical properties -Film thickness - Film morphology - Assembly kinetics

- No multiplexing Detection limit: femtomoles/cm2

QCM-D SAv b-GAGs Application : Quantification and characterization of

20 model surfaces presenting Glycosaminoglycans (GAGs)
0

-20 15 7 nm
12 nm

D (10 )
-40

-6
f (Hz)

10

-60
5 4 nm
sssssssssssssssssssssssssssssssssssssssss
-80 Au
0 6 nm 5 nm
-100
0 20 40 60

Time (min)

• Streptavidin (SAv) monolayer: rigid and dense monolayer.

• b-GAGs monolayer: soft and highly hydrated film.
• Control and characterization of the supramolecular presentation of HS chains.

Migliorini et al., Biomaterials, 2014, 35, 8903

Quartz Crystal Microbalance in liquids
Application: Discrimination of endo or exo mechanisms of an enzyme

Bouchet-Spinelli et al., Biosens. Bioelectron. 2013, 49, 290

Surface tension based detection at µcantilevers

Deflection Influence of the ionic

Static mode force and of the nature
Deflection of the µcantilevers due to of the media
modification of the surface entension
Cantilever lenght
M Guirardel, Thesis of the University of Toulouse, 2003 Poisson constant
Differential surface energy between the 2 faces
DNA detection at vibrated diamond cantilevers
HO O
C HO O -
HO O
C C

H2N

Detection of protonation according to surface energy modulation

Variation of resonance frequency according to the hybridization state
Frq increase
Bioelectronic: according
B- Molecular DNA probe
bioelectronic concentration
2. Enzymatic biosensors following by non-specific reorganisation 30
Bongrain et al, 2011, Langmuir, 27 12226
Ionic conductivity in nanopores

a-hémolysine

DNA sized proteic nanopores

Separation and counting of nucleosides
Akeson et al, Biophys. J. 76 1999 3227.

Evolution toward artificial nanopores

Aksimentiev et al, Biophys. J. 87 2004 2086.
Ionic conductivity in nanopores

Applications
Ionic conductivity in nanopores
Ionic conductivity in nanopores

Two pore guys,

Inc

http://twoporeguys.com/home/

20 x 30 nm nanopore in silicon
nitride
2 nanopore in series
Ionic conductivity in nanopores
Which transducer for better sensitivity?
 C-
Cell Chips
Cell chips, some generalities

Biochips :
-Emerging owing to microelectronics development
-Biochips : DNA, proteins, sugars & cells

Cells :
-Complex biological samples (blood, sera…)
-Specific investigation and application fields
-Sample : little population-
individual cells to unique cellules

Benefits :
-Reduction in sample volume
-Process automation (reliability/quality)
-Specific properties of this working scale
-Speed and reliability of analysis, cost and sometimes gains in sensitivity,
etc.
Cell chips: 2 main strategies
1) « Lab-On-A-Chip »

 Reduction and adaptation on experimental protocol of cell

biology utilized in the macroscopic scale

 Tend to analyse individual or unique cells

 Applications :
Cell handling
Cell separation
Rare cell searching
Cell lysis
Nucleic acids and proteins extraction
Particule labelling
Drug screening
…
Tegafur = oral prodrug of 5-fluorouracil (5-FU)

A micro cell culture analog with 3-D hydrogel culture of multiple cell lines
to assess metabolism-dependent cytotoxicity of anti-cancer drugs
Sung et al., LOC, 2009
« Lab-On-A-Chip » for fluid handling
Cell handling:
Fluids handling:

DeMello, LOC, 2008

Edd, LOC, 2008 Wang, X.B., et al., Anal Chem, 2000.

Séparation of monocytes versus LB
« Lab-On-A-Chip » for cell lysis

1-100 cells Quake SR

20nL reactor Nature Biotechnology 22, 435 - 439, 2004
A nanoliter-scale nucleic acid processor with parallel architecture
Cell chips: 2 main strategies
2) « Micro-array »

 Highly parrallelized assay (1 cell type / n conditions or n cell types / 1

condition). Possible adaptation for screening

 Historical and technical evolution ofclassical biochips (DNA, proteins…)

 Data collection (signal and/or images acquisition) and control analysis

(High data throughput to treat)

 Application to small cell populations or to individual cells

« Micro-array » as screening tool in genomics
Screening tools for functional genomics
Ziauddin, Nature, 2001

100 µm

(phospho-MAPK proteins)

 Highly parallelized gene transfection (192 genes + TAG V5 / HEK293T)

 Screening of cellular events: cascades of phosphorylation, apoptosis, cellular
adhesion
 Example of phophorylated genes adducts of the MAPK protein
 Intra-cellular location of the expressed adducts
« Micro-array » for infection monitoring
Lentivirus-infected cell microarrays (LICM)

Cells infected by lentivirus : over-expression of cDNA (GFP) or expression shRNA (Laminin

A/C)
 Screening of phosporylation cascades (signalisation using Tyrosine kinases), apoptose,
cellular adhesion
 Start-up : Akceli
Bailey, Nature Methods, 2006
« Micro-arrays » in phenotypic studies
Highly paralelized phenotypic studies

 Adherent proteins patterns(fibronectine)

 Monitoring of cell polarization
Centrosome nuclei vector 2D-tracing
Capture on identical patterns
 Monitoring of microtube nucleation and migration

Reconstructed trajectory of microtubes.

« Micro-array » for directed cell adhesion

 Reduction in the variability of the intra-cell organisation owing to the geometry control
of cell adhesive microenvironment
 Phenotypic variations of the cells automatically detected
 Application filed : adherent cells
 Screening tool
CELL CHIPS FOR CIRCULATING CELLS
Circulating cells such as lymphocytes are important cells in body inflammatory response.

Their activity is strongly involved in immune response to bacteria, virus, retrovirus

agressions…

The understanding on their chemical communication pathway is of the first importance

to treat many diseases including AIDs, Hepatitis C virus but also autoimmune response
(cancer)…
Immobilisation of circulating cells
Owing to the diversity of cell chemistry
Macroscopic cell partitioning : FACS

Surface immobilization:

LB LT

anti-LB IgG anti-LT IgG

Immobilized antibodies
2D cell organization
No limitation in type lines
Source : http://www.bdbeurope.com
Local electrochemical immobilization of antibodies

IgG
PBS, pH 7.4, overnight, 4°C
Pyrrole conjugated
IgG
2V ddp,
100ms SPrAM/CREAB group
aqueous
solution CEA Grenoble
free pyrrole or unsoluble polypyrrole polymer
conjugated pyrrole deposited on the anode

CE

WE
Cell capture imaging using SPRi

PUMP
Ab
biochip sample
surface

100 m
CCD
LED camera

mixed cell
CD 19
CD90 pop. inj.
T Lymphocyte
pure cell
-LS102.9 cells (300 µL, 105 cells.mL-1) at t= 0
B Lymphocyte minutes
pop. inj.
-DMEM flush (for 12 minutes) at t= 15
I-Ab CD3 minutes
-mixed populations of LS102.9 and 13G7
cells (10:1) (300 µL, 105 cells.mL-1 and 104
cells.mL-1 respectively) at t= 27 minutes
(incubation for 15 minutes).

E. Suraniti, et al., Lab on a Chip, 2007

Cell secretion characterization

 Immune system is highly complex

 Interest in secretion monitoring:

Immunomonitoring
Toxo et HBV immunity
Allergical desensibilisation

 State of the art techniques:

ELISA
ICS
ELISPOT
Myltenyi assay (4Ac)

end point detection

labelling steps are required
limited throughput
Cell chip fabrication and reading
Cell culture at biochip surface
CytC 20µM

Hybridoma cells suspension HEL 5µM

HEL 10µM

HEL 20µM
sample chamber
secreted IgG
specific antigen
biochip surface poly pyrrole spot

LED
CCD
camera

SPR
Milgram et al., Biosensors & Bioelectronics, 2010
Why a so fast response?

SPR imaging DURING cell culture

cell culture, protein secretion

ELISA assay
- Dilution
- Labeling
- Washing step
- Enzymatic coloration
- Readout 0,25pmoles in 1 h for 250000 cells  about
2 mM in 10min, at the unique cell scale
(Ø≈16µm) !!!

Quantification in solution Confined space detection

The fisherman paradox: trapping …and release a target

 Advanced applications (proteome-transcriptome) or release of selected cells for

further culture

 Antigen antibody interactions are strong and complex: no generic recipe to release
antigen (cell) from antibody capture

 Cells are chemically sensitive and mechanically fragile

 Biochips are parallelized microsystems : different cell types may be immobilised

requires a sequential and directed release
Specific chemistry for cell release: the enzyme route

Enzymatic cleavage of a specific site: Which enzyme???

Restriction enzyme:
-high specificity
-large library
-high activity
-no parasitic activity

How to change a DNA chip into an antibody chip?

DNA-Ab
hybrid
DNA*

restriction
site

BIOCHIP TARGET ENZYME DE

FUNCTION- CAPTURE RESTRICTION
NALISATION
BIOCHIP RE-
DNA GENERATION
biochip
Cell capture and release monitoring using SPRi

ENZYMATIC
B lymphocyte CLEAVAGE

anti-CD19
Ab
restriction site

Capture
probe
prism

18/0
1/20
Physical cell release using local heating

LASER
Source

Local heating through local

plasmon generation

Semi-absorbant slide

CCD
LED LASER light Caméra
absorber

 Experimental setup development

 First test using ds-DNA-biotin-Streptavidin-PS beads
One spot-one biological object: how to create artificial cell arrays
Polypyrrole electrodepositio using bioplumes

spot size = 3-15µm - lymphocyte = 10µm

electrochemistry run in attoliters

Roupioz et al., Small, 2009, 5,13, 1493-1497

CONCLUSION

Biochips have been extensively studied and developed to enable large-scale genomics,
proteomics, even cellomics...

This course is absolutely not exhaustive but hope you are convinced now.