cost effective electrochemical sensors using …...btm (bone turnover marker) sensors 29 • need...

PUBLIC

COST EFFECTIVE ELECTROCHEMICAL SENSORS USING

ADAPTED PCB TECHNOLOGIES FOR THE DETECTION OF

BIOMARKERS

2ND WORKSHOP ON PCB BIO-MEMS

MAY 31, 2017, MERIDEN, UK

JAN VANFLETEREN, PATRICIA KHASHAYAR, LOTHAR MADER, PIETRO SALVO

CENTER FOR MICROSYSTEMS TECHNOLOGY, IMEC, ZWIJNAARDE, BELGIUM

WWW.CMST.BE

JAN.VANFLETEREN@IMEC.BE

CONTENTS

CMST introduction

Electrochemistry for biomolecules detection

FP7-MIRACLE project : DNA detection

Bone Turnover Marker (BTM) sensors

H2020 PoCOsteo project

Conclusions and acknowledgement

CONTENTS

CMST introduction

CMST: DESIGN & TECHNOLOGY OF MICROSYSTEMS

ORGANIZATIONAL STRUCTURE

Faculty of Engineering and Architecture

Department of Electronics and Information Systems

Smart Systems and Energy Technology

Body Area Networks

Personnel Equipment

Personnel Infrastructure

Equipment

CMST INTRODUCTION – ULTRA THIN CHIP PACKAGE

CMST introduction – PCB based stretchable circuits

CMST INTRODUCTION – CELL CULTURING AND MICROFLUIDICS

Integration of electrical functions in microfluidics

Porous membrane integration

seeding

Osmotic pump

Chip in PDMS microfluidics

(LED/photodiode aray)

Soft polymer based bioreactors

CONTENTS

CMST introduction

ELECTROCHEMISTRY FOR BIOMOLECULES DETECTION

Principle :

• Modulation of an electrical current (ion

current decrease / charge transfer

(current generation) by binding

biomolecules to a conducting

(working) electrode

• Current level is determined by the

concentration of bound biomolecules

• Selective binding necessary

Working electrode

Cross linking molecules

(1) Antibody or (2) DNA fragment

Biomolecule to detect : (1) protein antigen, or

(2) DNA fragment

• We use redox probes (e.g. K3[Fe(CN)6], PBS,...)for (ionic) current generation

• For the protein/antibody case : the higher the protein concentration, the more proteins will bind to the antibodies, the more difficult the ions of the redox probe will reach the working electrode, the lower the measured electrical current will be.

• For the DNA case : attachment of first DNA current decrease (similar as protein/AB); attachment of second DNA fragment generates a charge transfer and a current

• Required : starting substrate with conductive electrodes (Au)

• Technology options :

• Thin-film (e.g. sputtered) (on glass or ceramic substrate)

• Printed (screen, stencil, inkjet)(on glass or ceramic substrate)

• PCB based (on FR4 or PI/PET flex substrate)

• Thin-film (fine patterns, <10micron) and printed (additive, more cost

effective) are standard; can PCB offer an alternative ?

CONTENTS

CMST introduction

FP7-MIRACLE

• First use case : EU-FP7-MIRACLE (2010-2014) : “Magnetic Isolation and

moleculaR Analysis of single CircuLating and disseminated tumor cElls on

chip”

• Genetic analysis using electrochemical sensor, responsible partner : URV

(Taragona, Spain)

• At the beginning of the project URV used thin-film-on-glass electrodes

imec suggested switch to PCB, developed the technology, and delivered

PCB sensor array samples to URV.

FP7-MIRACLE

Design:

• 64 working electrodes

• 25mm x 25mm substrate

• Only active electrode surfaces exposed to biofluid, other surfaces (interconnections) isolated

FP7-MIRACLE

• Sustrates :

• FR4 rigid (1.2mm FR4 + 18µm Cu)

• PI flex (50µm PI + 18µm Cu)

• PET flex (125µm PET + 3µm Cu)

• Isolation layers :

• SU8 (negative photoresist)

• AZ4562 (positive photoresist)

• Screen printable solder mask

Process (key steps): 1. Preparation of boards (micro-

etching) 2. Lithography + etching

1. Spinning resist (AZ4562 at 2000 rpm for 60 s)

2. Illumination (UV exposure)

3. Developing (AZ4562 developer for 2 min)

4. Spray etching (CuCl2 / HCl)

5. Stripping (AZ400K developer for 2 min).

3. Electroless NiAu plating (hypophosphite bath)

4. Deposition of passivation layer

FP7-MIRACLE

Polyimide flex after

electroless NiAu

Finished substrate :

FR4 + solder mask

FP7-MIRACLE

• Conclusions :

• Solder mask works best as passivation

• All 3 substrate types can be used, preference for FR4 and PI

• Electroless Ni/Au : 5µm Ni (can be modified) + “flash” Au (limited to <1µm)

not fully biocompatible, probably because of nickel diffusion on the surface

through thin Au adapted process necessary with thicker (electroplated) Au

FP7-MIRACLE

Adapted process : replace electroless NiAu by electroplated Ni/Au (Enthone chemistry)

1. Nickel deposition (Lectronic 10-03), 5 µm, on copper

2. Strike-gold (Aurobond), 200 nm, used to prevent nickel diffusion and improve gold adhesion

3. Gold deposition (Neutronex 309), 3 and 5 µm.

Design change : Cu ring (shorting all electrodes for electroplating), to be removed mechanically

Cathode Plating Clamping area

FP7-MIRACLE

Cross section : 5 µm Ni / 5 µm Au

FP7-MIRACLE

Finished sensor array (FR4 / solder mask passivation)

FP7-MIRACLE

• Roughness of plated NiAu increases sensitivity

• No difference between 3 and 5 µm Au

Cyclic voltammogram recorded in 0.5 M H2SO4, 100 mV s−1 (electrode cleaning) (oxidation @ 1.1V and reduction @ 0.7V visible)

5 mM K3Fe(CN6) clean, mercaptoundodecanoic modified (MUA) and DNA modified (DNA) 3 μm thick gold electrodes)

FP7-MIRACLE

Selective detection of breast cancer markers : 5 nM of TACSTD1 target at the electrodes modified with either complementary TACSTD1 probe, or non-complementary GRP, MYC, SCGB2A1, SCGB2A2, TOP2A

Response from TACSTD1 specific and DT (Backfiller) sensors for different concentrations of TACSTD1 target

CONTENTS

CMST introduction

BTM (BONE TURNOVER MARKER) SENSORS

Bone turnover is essential for bone health. It is a process characterized by 2

tightly coupled activities:

First being torn down (bone resorption) and then being rebuilt (bone formation)

Formation Resorption

Remodeling Balance

Stable Bone Mass

After middle age, bone loss occurs as resorption

exceeds formation…

Formation

Resorption

Negative Remodeling Balance

Bone Mass decrease

.... Potentially leading to osteoporosis :

• Fracture is a common but drastic osteoporosis complication imposing a

heavy burden on both the society and family

Current diagnostics : not suitable for preventive screening, nor for treatment monitoring

BMD (bone mineral density) : X-ray based,

detection only when BMD significantly decreased (bone restauration impossible),

no information on cause of osteoporosis (bone resorption / formation);

2 years interval between tests needed to see significant changes in monitoring treatment

ECLIA (Electrochemiluminescence Immunoassay) & ELISA (Enzyme-linked immunosorbent assay) : expensive,

not available everywhere,

skilled technician needed;

long waiting time in centralized laboratories

(GE Lunar)

(TZD Technology)

• Need for a simple, yet accurate PoC device for osteoporosis prevention,

diagnosis, treatment follow-up

• Development of such a device was done in the frame of a joint PhD (P.

Khashayar, Ghent University and Tehran University, Iran) (2014-2017).

• Aim : development an electrochemical sensor + microfluidic chip for at

least 1 marker for bone formation (OC (osteocalcin), or P1NP) and 1

marker for bone resorption (CTX)

Implementation during PhD work : 1. Glass

2. sputtered / photolithography / wet etching defined 50nm TiW/ 100nm Au thin film

3. SU8 passivation

• Au NP (nanoparticles) deposited through ECV (electrodeposition using cyclic voltammetry)

• GSH = gluthathione

• Ab’s (antibodies) immobilized on GSH through sulfo-NHS (N-hydroxysuccinimide) crosslinkers

Au NP’s : considerable increase of roughness and total active area of the electrode surface

• Microfluidic device build-up : 1. Glass + thin film electrodes

2. Laser structured double sided tape (3M-9965 biocompatible tape)

3. Top plate (COC) with access holes

Tape partially cut to facilitate the handling, aligning and assembling process

Final assembly

Electrical interconnection

Fluidic interconnection

Tape thickness = channel height = 85...90

Measurements on real serum samples : good agreement between our PoC tool and “golden standard” ECLIA measurements (executed at UGent University Hospital for both types of markers (osteocalcin and CTX) in relevant concentrations

• Replacement of glass by PCB : 150nm metallisation (TiW/Au) replaced by 17μm Cu + 5 μm Ni + 5 μm Au

• Main differences with MIRACLE dedicated PCB fabrication process :

• Now start from commercially purchased PCB + Cu pattern; perform Ni/Au plating steps in-house

• New Au plating bath (proprietary, composed from commercially available ingredients)

• Non-uniformity in Au plating results cleanliness (e.g. rinsing steps) in commercial PCB manufacturing perhaps insufficient for our back-end processing ? switch to full in-house PCB production

Clean Dirty

PCB + 3M tape + rigid 1mm COC top plate leakage

PCB + 3M tape + thin (135 μm) COC (Topas) top foil no

leakage !

CONTENTS

CMST introduction

H2020-POCOSTEO PROJECT

• H2020-RIA, Topic NMBP-13-2017 : Cross-cutting KETs for

diagnostics at the point-of-care

• Aim : development, clinical validation and

preparation for commercialisation of a Point-of-Care

tool for bone disease (a.o. osteoporosis) prevention,

detection and treatment.

• Budget : 4Meuro

• Duration : 4 years

• Estimated start : October 1, 2017

Partners : • Ghent University (UGent, Belgium) : coordinator, development of

proteomic sensor

• Universitat Rovira i Virgili (URV, Spain) : development of genomic

sensor

• Fraunhofer IMM (Germany) : microfluidics research

• microLIQUID (µLIQ, Spain) : microfluidics production (SME)

• Labman (UK) : PoC tool production (SME)

• Medical University of Graz (MUG, Austria) : clinical validation

• Endocrinology and Metabolism Research Institute (EMRI, Iran) : clinical validation

• Fundico (Belgium) : administrative coordination

Sensor development challenges : • Increase sensor shelf life to > 6 – 12 months (proper packaging,

chemical electrode surface modification,…)

• Implementation in cost effective technologies : PCB, printing

• Transfer to an industrial partner – microLIQUID has no PCB manufacturing capabilities

CONTENTS

CMST introduction

CONCLUSIONS

• PCB technology is an alternative for the cost effective fabrication of electrochemistry based biosensors, with printing as a potential competing technology

• Feasibility of PCB biosensors was demonstrated for the selective detection of cancer markers (FP7-MIRACLE)

• Transfer to industrial environment of PCB biosensor substrate production might require upgrade of production standards (cleanliness, etc.)

• Potential wide application area, our next target application will be osteoporosis management (H2020-PoCOsteo)

ACKNOWLEDGEMENT

Funding projects : • EC-FP7-MIRACLE (Grant Agreement Number 257743)

• EC-H2020-PoCOsteo (Grant Agreement Number 767325)

Questions?

Thank you !

PUBLIC

cost effective electrochemical sensors using …...btm (bone turnover marker) sensors 29 • need...

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