Supplementary information

Microchip design

Excitation electronics The schematics of the HVPS could be found below (Figure S2).

a)

Fig. S1 a) Picture of the micro-milled mould. b) Picture of the final microfluidic chip, after

bonding of the PDMS replica on the patterned glass slide. c) Picture of the microfluidic chip and

the HVPS.

b)

c)

2mm 3mm

Electronic Supplementary Material (ESI) for Analyst.This journal is © The Royal Society of Chemistry 2017

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A A

B B

C C

D D

E E

Date: 26 feb 2014KiCad E.D.A.

Rev: 1.0Size: A3Id: 1/1

Title: Module excitation HT

File: gene_HT.schSheet: /

KENIUM

GND1

VCC2

VLCD3

RS4

R/W5

CS6

D07

D18

D29

D310

D411

D512

D613

D714

LED+ 15

LED- 16

S1

FDCC2004C FLYYBW-51LK

VD

D1

RA5 2

RA4 3

MCLR/RA3 4

RC55

RC46

RC37

RC28

RC19

RC010

RA2 11

RA1 12

RA0 13

VS

S14

U7

PIC16F1825

CLK1

Din2

CS3

Dout4

GN

D5

RE

F6

Vout 7

VC

C8

U10LTC1452

DIP8

Vbus1

D-2

D+3

GND4

Shield_15

Shield_26

J1

US

B

RA5/AN4/SS/HLVDin/C2OUT 7

MCLR/Vpp/RE31RA0/AN0 2

RA1/AN1 3

RA2/AN2/Vref-/CVref 4

RA3/AN3/Vref+ 5

RA4/T0CKI/C1OUT/RCV 6

RE0/AN5/CK1SSP 8

RE1/AN6/CK2SPP 9

RE2/AN7/OESSP 10

RD1/SPP1 20

RD7/SPP7/P1D 30

RB7/KBI3/PGD 40

VDD11

RD2/SPP2 21

VSS31

VSS12

RD3/SPP3 22

VDD32

OSC1/CLKI13

RC4/D-/VM 23

RB0/AN12/INT0/FLT0/SDI/SDA 33

OSC2/CLKO/RA614

RC5/D+/VP 24

RB1/AN10/INT1/SCK/SCL 34

RC0/T1OSO/T13CKI 15

RC6/TX/CK 25

RB2/AN8/INT2/VMO 35

RC1/T1OSI/CCP2/UOE 16

RC7/RX/DT/SDO 26

RB3/AN9/CCP2/VPO 36

RC2/CCP1/P1A 17

RD4/SPP4 27

RB4/AN11/KBI0/CSSPP 37

Vusb 18

RD5/SPP5/P1B 28

RB5/KBI1/PGM 38

RD0/SPP0 19

RD6/SPP6/P1C 29

RB6/KBI2/PGC 39

U4

PIC18F4455-DIP40

11 2 2

33 4 4

55 6 6

77 8 8

99 10 10

P1

HE10-10

1

23

CON1

24V

1

2

ON12 3

4SW3

SW-DIP2

+5V R4

22K

SW1

SW_PUSH

+5V

D1

1N41

48

+ C1

1µF

+5V

C7

10nF

X1

20MHz

C4

15pF

C6

15pF

+5V

1

2

3

RV110K

C11

220nF

VI VO

GN

D

U2

TSR-2450+5V

PWR_FLAG

PWR_FLAG

+ C5

10µF

SCL>

SDO>

SDI<

+5V

C16

10nF

+5V

C19

10nF

SS>

RAZ

RAZ

INT-MES<

MESI1

SW2

SW_PUSH

R1

10K

+5V

PB_ON

PB_ONLEDRLEDV

LEDR

LEDV

1 2D2

SRD

1 2D3

SGD

R23.3K

R31K

+5V

V+

1

IL3

CA

4

CA

5

6-20

+21

V-

24

U6APA343

-8

+9

V-

12V

+13

IL15

CA

16

CA

17

18

U6BPA343

CLK1

Din2

CS3

Dout4

GN

D5

RE

F6

Vout 7

VC

C8

U9

LTC1451

DIP8

+5V

C17

10nFE 1

C 2

B3

Q1

BD237

+24V

+24V+24V

R7

332K

1%

PWR_FLAG

+IN1

-IN2

+OUT 3

-OUT 4

CT 5

ER

R6

LOG

5V7

DIS

8

U3

FS05CT

R19100

R20100

P2 HT+

P3 HT-

1

2

3R

V2

10K

+5V

R2310K

+5V

run

debug

prog

ON ON

ON -

--

SW2 1 2

SS>

SDO>

SDI<

SCL>

INT-MES<

R9

332K

1%

R8

13.3

K 1%

R10

13.3

K 1%

DIS-HT

DIS-HT

CLKLDINLCSL

CLKLDINLCSL

R15

470

+5V

R510K

1 2D4

SBC

R6330

+5V

C2

100nF

+5V

+130V

-130V

level out0-2.4V

CAP+2

GN

D3

CAP-4VOUT 5

LV 6OSC7

V+

8

U5

ICL7660

+5V

C9

10nF

+ C8

10µF

+C10

10µF

-5V

-5V

+5V C21

10nF

R26

10K

1%

R2410K

1%

R2510K

1%

R2710K

1%

1-2

+3

V-

4V

+8

U12A

LM1458

1-2

+3

V-

4V

+8

U1A

LM358 C22

10nF

C3

10nF

C13

10nF

C14

10nF

C15

10nF

+HT

+HT

+HT

-HT -HT

-HT

R131K

1%

R141K

1%

R1747.5K

1%

4.8V cc

R1647.5K

1%

R121K

1%

R111K

1%

C12

10nF

+ +

- -

1

2

3

4 5

6

7

8

U11 HCPL-7800

R22301

1%

+Vin1

-Vin2-Vout 5

+Vout 7

U8 IV0505S+5V

+5V

+5

-6

7

U1B

LM358

C20

10nF

+5

-6

7

U12B

LM1458

MESI1

C18

10nF

R21

470

SIG1 GND1

1 2JP1

JUMPER

E 1

B2

C 3

Q2

JE9012

E 1

B2

C 3

Q3

JE9013

X28MHz

C23

15pF

C24

15pF

CS1

SCK2

SDI3

VS

S4

PA 5

PW 6

PB 7

VD

D8

U14

MCP4151-104

+5V

C25

10nF

CS

SCL>CS

SDO>

MESI2

MESI2

SYNC

GND

test1

R29 39

C28

10nF

45U16B

4050

23U16A

4050

123 P

4C

ON

N_3 CLK

6 7

U16C

4050

9 10U16D

4050

11 12U16E

4050

14 15U16F

4050

+5V +5V

-5V

+5V C27

10nF

R30

10K

1%

R1810K

1%

R2810K

1%

R3110K

1%

1-2

+3

V-

4V

+8

U15A

LM1458C29

10nF

CS1

SCK2

SDI3

VS

S4

PA 5

PW 6

PB 7

VD

D8

U13

MCP4151-104

+5V

C26

10nF

SCL>CS

SDO>+5

-6

7

U15B

LM1458

pot. signal

pot. ref

100K

100K

74HC4050portes non utilisées

signal0-4.8V max

Oligonucleotide sequences

List of the oligonucleotides used for SDA-HRCA. Underlined sequences with the same colour are identical. Sequences with the same colour

are complementary. Nicking sequences are in italic and the nicked bond is bolded. Phos- indicates a 5’ phosphorylation and * indicates a

phosphorothiate bond. N is a random nucleotide.

Name Length Sequence

ldh1 165nt 5'-AGGTAATGGTGCAGTAGGTTCAAGCTACGCATTTTCATTAGTGAACCAAAGCATTGTTGATGAATTAGT CATCATTGATTTAGACACTGAAAAAGTTCGAGGAGATGTTATGGATTTAAAACATGCCACACCATATTCTCCAACAACAGTTCGTGTGAAAGCTGG

mecA 169nt 5’-TAATAGCAATACAATCGCACATACATTAATAGAGAAAAAGAAAAAAGATGGCAAAGATATTCAACTAA CTATTGATGCTAAAGTTCAAAAGAGTATTTATAACAACATGAAAAATGATTATGGCTCAGGTACTGCTATCCACCCTCAAACAGGTGAATTATTAGCACTT

Extension Primer 1 45nt 5'-GCATAATACTACCAGTCTCCTCAGCAAGCTACGCATTTTCATTAG

Extension Primer 2 44nt 5'-TAGAATAGTCGCATACTTCCTCAGCCATAACATCTCCTCGAACT

Bump Primer 1 19nt 5'-AGGTAATGGTGCAGTAGGT

Bump Primer 2 17nt 5'-CCAGCTTTCACACGAAC

RCA Padlock 90nt 5'-Phos-AGTCATCATTGATTTNNNNNNNNNNNNATCGACTGAGTGCTANNNNNNNNNNTAAGCGTTT CCCAAANNNNNNNNNNTTGTTGATGAATT

HRCA Primer 1 15nt 5'-ATCGACTGAGTGC*T*A

HRCA Primer 2 15nt 5'-TTTGGGAAACGCT*T*A

Signal acquisition and wavelet treatment Wavelet treatment was performed using the wavelet package in Matlab.

By trial and error, we validated the use of the Bior2.2 wavelet shape. In order to scan the complete conductometric signal, a collection of

wavelets 𝜑𝑗,𝑘 were generated from the mother-wavelet 𝜑.

𝜑𝑗,𝑘(𝑡) = 2−𝑗

2⁄ 𝜑(2 − 𝑗 𝑡 − 𝑘)

With 𝑗, 𝑘 ∈ ℕ, where 𝑘 controlled the temporal position of the wavelet and 𝑗 the spatial width and amplitude of the wavelet. Discrete

coefficients were then obtained by convolution with the temporal signal 𝑥(𝑡) to treat:

𝑑𝑥(𝑗, 𝑘) = ∫ 𝑥(𝑢)𝜑𝑗,𝑘∗ (𝑢)𝑑𝑢

Each collection of coefficients 𝑑𝑥(𝑗, 𝑘) with a fixed spatial width 𝑗 reflects the contribution of peaks with a similar width to the wavelet

𝜑𝑗,𝑘. This transformation could be inverted:

𝑥(𝑡) = ∑ 𝑑𝑥(𝑗, 𝑘)�̇�𝑗,𝑘(𝑡)

𝑘,𝑗

Where �̇�𝑗,𝑘 is the dual wavelet of 𝜑𝑗,𝑘. A detail signal 𝑆𝑗(𝑡) containing only the contribution of these peaks can be reconstructed using the

following formula:

𝑆𝑗 (𝑡) = ∑ 𝑑𝑥(𝑗, 𝑘)�̇�𝑗,𝑘(𝑡)

𝑘

The conductometric signal was then divided between several details signals 𝑆𝑗(𝑡) as seen on Figure S4:

The EHDA-related contributions were mainly distributed between the 2nd

, 3rd

, 4th

and 5th

detail signals, while the electronic noise was

present in the 1st

detail signal and the slow baseline shifts was recovered above the 6th

detail signal. A filtered signal 𝑥𝑤(𝑡) was thus

reconstructed with the physically relevant detail signals only:

𝑥𝑤(𝑡) = ∑ 𝑆𝑖(𝑡)

𝑖=5

𝑖=2

Further treatments were needed to remove transient artefacts in the wavelet-treated signal. Each measurement was divided into 10

segments 𝑥𝑤(𝑡𝑠 → 𝑡𝑠+1). To quantify both the quantity and the amplitude of the filtered peaks, we calculate the average of the absolute

value of the filtered signal. A partial quantification parameter for the segment 𝑠 is:

𝑄𝑠 = ⟨|𝑥𝑤(𝑡𝑠 → 𝑡𝑠+1)|⟩

The quantification parameter for the whole filtered signal is:

𝑄 = ⟨|𝑥𝑤(𝑡0 → 𝑡𝑓)|⟩

Fig. S3 From top to bottom: raw signal (in mV) from a

conductometric measurement of Lambda-DNA aggregation

(blue). Detail signals associated: 𝑆2 (𝑡), 𝑆3 (𝑡), 𝑆4 (𝑡), 𝑆5 (𝑡)

(black). Reconstructed signal 𝑥𝑤(𝑡) (red)

If a segment had a quantification parameter higher than 12% (value validated with optical controls) of 𝑄, it would be removed and the

quantification parameter would be updated to:

𝑄 = ⟨|𝑥𝑤(𝑡0 → 𝑡𝑠 & 𝑡𝑠+1 → 𝑡𝑓)|⟩

The algorithm was looped until no segment was removed and the previous quantification parameter was kept.

Gel electrophoresis protocol The amplification products were analyzed by agarose gel electrophoresis, concentration 4% in a TAE 1X buffer. The gel was premixed with a final concentration of 1X SyBr Gold before casting. 0.5µl of SDA sample and 25bp ladder (from Invitrogen) were mixed with 1µl of Blue/Orange 6X loading dye (from Promega) and 4.5µl of TAE buffer. 5µl of mix was deposited in the gel wells. Electrophoresis was performed during 1h at 100V. Imaging of the gel was done in an Amersham 600 Imager. SDA and HRCA scheme

Extension Primers

Nicking enzyme

Fig. S4 SDA scheme. 3’ ends are indicated with arrows. a) &

e) Primer-target hybridization. b) & f) Primer-target

elongation. c) & g) Nicking. d) & h) Elongation from the

downstream strand and strand-displacement of the

upstream strand.

Polymerase with a strand-displacement

activity

a)

c)

d)

e)

b)

f)

g)

h)

Polymerase with a strand-displacement activity

DNA Ligase

HRCA primers

b) Ligation

c) RCA

d) HRCA

a) Padlock probe hydridization

Fig. S5 HRCA scheme. 3’ ends are indicated with arrows. a)

Hybridization of the RCA Padlock probe with the SDA single-

stranded amplicon. b) Ligation of the RCA Padlock probe

into a DNA circle. c) Priming of the circle with a primer, and

displacement of the SDA amplicon. d) Branching of the RCA

multimer with the primers