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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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