Nano Inspired Biomedicine Laboratory 1 Department of Surgical, Oncological and Gastroenterological Sciences, University of Padua, Italy. 2 Istituto di Ricerca Pediatrica- Città della Speranza, Padova, Italy.

1. Nuove tecnologie per lo studio del DNA tumorale libero circolante
M. Agostini1,2
1 Department of Surgical, Oncological and Gastroenterological Sciences, University of Padua, Italy.
2 Istituto di Ricerca Pediatrica- Città della Speranza, Padova, Italy.
Nano Inspired Biomedicine LaboratoryNano Inspired Biomedicine Laboratory

2. cfDNA quantification methods
Spectrocopic techniques:
• Spectrophotometry: NanoDrop (Thermo Fisher Scientific)
• Colorimetry: DNA DipStickTM
Kit (Invitrogen)
• Fluorimetry (PicoGreen® Dye, SYBR® Gold Dye, Hoechst 33258 assay)
Fast and easy detection of dsDNA
Other techniques:
• Plasmonic nanosensor with inverse sensitivity (DNA
concentration-dependent gold nanorod aggregation)
Pallares RM et al., 2015

3. Schwarzenbach et al. 2012
NATURE REVIEWS | CANCER

4. Other techniques:
• Atomic Force Microscopy (AFM) provides direct visualization
and measurement of individual DNA fragments
Separation techniques:
• Capillary zone electrophoresis (CZE) with laser-induced
fluorescence detection (LIF)
• Microarray dielectrophoretic (DEP) device (Biological
Dynamics)
Fragments evaluation of tumoral DNA
Pang D et al, 2015
Sonnenberg A et al, 2014
Zang P et al, 2004
SYBERGold inetercalating
dye, fluorescein as
internal standard)

5. Molecular biology techniques:
• Quantitative Real Time-PCR
Single gene:
APP;
ACTB;
b-globin;
b-2-microglobulin;
GAPDH;
RNAse P;
hTERT
branched-DNA:
Alu sequences;
B1 sequence in mouse (Alu
equivalent)
Genomic Interspersed Repeats:
LINE-based;
Alu-based
SYBR chemistry
TaqMan assay
SYBR chemistry
Total cfDNA and tumoral DNA detection
Zang L et al, 2010

7.  Evaluation of cell-free DNA as a biomarker for pancreatic malignancies. Int J Biol Markers. 2015.
 Circulating cell-free DNA, SLC5A8 and SLC26A4 hypermethylation, BRAF(V600E): A non-invasive tool panel for
early detection of thyroid cancer. Biomed Pharmacother. 2013
 Circulating cell-free DNA: a promising marker of regional lymphonode metastasis in breast cancer patients.
Cancer Biomark. 2012.
 Circulating cell-free DNA: a promising marker of pathologic tumor response in rectal cancer patients receiving
preoperative chemoradiotherapy. Ann Surg Oncol. 2011

9. ALU 247 (ng/ml)
0 20 40 60 80 100
100
80
60
40
20
0
100-Specificity
Sensitivity
AUC
(95%CI)
p-value
Cut-off
(ng/ml)
Sensitivity
(95%CI)
Specificity
(95%CI)
ALU244
0.97 (0.9-1.0) 0.0001 >2.0065 94.0 (85.4-98.3) 100.0 (89.9-100.0)
100000
10000
1000
100
10
1
ALU115ng/ml
TRG 1-2 TRG 3-4-5
100000
10000
1000
100
10
1
ALU247ng/ml
TRG 1-2 TRG 3-4-5
p.Value<<0,05p.Value<<0,05
Alu83Alu83
Alu244Alu244
Integrity of CfDNA Alu247/Alu115Integrity of CfDNA Alu247/Alu115
A
B
C

10. Who we are
2
• Approximately 43,600employees worldwide
• Approximately 8,700 people working in R&D
• 11 manufacturing sites
• Over 75 sales & marketing offices
• A global semiconductor leader
• 2014 revenues of $7.40B
Front-End
Back-End
Research & Development
Main Sales & Marketing
Listed on New York Stock Exchange, Euronext Paris
and Borsa Italiana (Milan)

11. ST MEMS products
Printer heads
11
Motion sensors
Active microphones
Drug delivery pumps
Lab on Chips

12. Trends of microelectronics
12
Miniaturization
Price of 1 Mbit of Memory5 000 €
1977
400 €
1981
120 €
1984
30 €
1987
5 €
1990
0.05 €
2000
75 000 €
1973
0.5 €
1995
0.0005 €
2015
Lower cost
Ease of use

13. Q3-Plus: a new system partition
• Low instrument cost:
– No need for huge initial
investment
• Pay per use:
– No need to perform a
minimal number of tests
to be cost effective
• Simpler use:
– The disposable is pre-
functionalized and has
the highest industry
quality standards
– Less trained personnel
13
Instrument Disposable
Today
New vision

14. Q3-Plus for real-time PCR: key facts
• The Q3-Plus system is a compact platform which permits
analysis of DNA-RNA through real-time PCR, for applications
ranging from medical diagnostics to environmental control
and food production
• It is designed for easy use by unskilled personnel as a point-
of-care system
• Compared with standard lab instrumentation, the lower
price of Q3-Plus eliminates the need to perform a batch of
tests in order to be cost effective.
14

15. Q3-Plus system partition
15
InstrumentSoftware
Biological content
Primers
Enzymes
Targets
Protocols
(from ST partners)
Disposable cartridge

16. Cartridge: overview
• Single-use disposable device
• All the reagents can be included at manufacturing time
• Room temperature storage
• Shelf life > 1 year
• 6 reaction chambers @ 5-20 µl each
• Very precise, in situ thermal control
• Suitable for many application fields:
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• Infectious diseases
• Oncology
• Genotyping
• Agro-Food

17. Instrument: overview (1)
• Independent modular system that
thermally drives the cartridge and
houses the illumination and detection
components all-in-one
• CE-marked, complying with the
2004/108/EC and 2006/95/EC EU
Directives.
17

18. Instrument: overview (2)
• Weight: 300 g
• Dimensions: 14 x 7 x 8.5 cm
• 12 V, 1.2 A power supply
• USB connection to PC
• Optional battery operation
and wireless connection
18
• Thermal control, 0.1°C resolution
• Easy plug for cartridge insertion
• LEDs and filters for fluorescence excitation
• 4 filtered CMOS micro-cameras for
fluorescence detection: 4 channels

19. Q3-Plus system specs (1)
Cartridge
 Appearance: silicon microchip with mounted plastic frame
 Size: 46.9 L x 21.9 W x 6.2 H (mm)
 Reaction site thermal control: integrated sensor and heaters
 PCR reactor volume: 5-20 µl @ 6 reaction chambers
 Liquid loading system: micropipette
 PCR reactor sealing system: Self-seal based on phase-change paraffin wax
Instrument
 Size: 14 L x 7 W x 8.5 H (cm)
 Weight: 300 g
 Power requirements: 12 V DC, 1.2 A
19

20. Q3-Plus system specs (2)
Performance parameters
 Temperature control accuracy: ± 0.2 °C
 Heating rate: 10 °C/s
 Cooling rate: 4 °C/s
 Temperature resolution: 0.1 °C
Biochemistry & optics
 Nucleic acid type: DNA/RNA
 4 independent fluorescence channels
 Labeling dyes: FAM™/SYBR®
Green/EvaGreen®
(emission range 510-540 nm),
VIC®
/HEX™/TET™/JOE™/MAX™ (540-570 nm), NED™ (560-590 nm), ROX™/Texas Red (590-620
nm)
 Detection chemistry: DNA-intercalating dyes (e.g. SYBR®
Green/EvaGreen®
), probes (TaqMan®
MGB or TAMRA™
, Scorpions®
, …)
20

21. “Open” control software v.3
• General purpose, easy-to-use interface to develop new applications
• Fully customizable temperatures, reaction times, optical parameters
• Robust image analysis
• Generic PCR data analysis (fitting of raw data, CT calculation, absolute
quantification)
• Melting analysis.
21

22. “Open” control software v.4
• Capability to run multiple instruments from a single instance
22
• Unlimited number of
instruments can be added
to work simultaneously
• Dashboard, providing the
status of each instrument
• Dedicated pop-up
windows for detailed
control of each
instrument.

23. Std 4 Std 5 Std 6 Std 7 Std 8
blank
Std 2 Std 3
ALU 83
Delta Rn vs Cycle
blank 27,5 Std7 25,8 Std6 22,8
Std5 23,3 Std4 17,1 std3 15,4
blank 30,2 Std7 26,9 Std6 27,6
Std5 19,9 Std4 18,1 Std3 18,9
1000 pg/μl std2 9,1
100 pg/μl std3 13,3
10 pg/μl std 4 16,4
1 pg/μl std 5 19,2
0,1 pg/μl std 6 23,0
0,01 pg/μl std 7 25,5
0,001 pg/μl std 8 27,8
blank 31,7

24. Delta Rn vs Cycle
ALU 244
Std 4 Std 5 Std 6 Std 7 Std 8
blank
Std 2 Std 3
H20 Neg Std7 27 Std6 29
Std5 31,6 Std4 17,3 Std3 18,5
H20 31,1 Std7 23,9 Std6 28,5
Std5 32 Std4 17 Std3 12,9
Delta Rn vs Cycle
1000 pg/μl std2 11,8
100 pg/μl std3 14,8
10 pg/μl std 4 18,5
1 pg/μl std 5 22,1
0,1 pg/μl std 6 25,4
0,01 pg/μl std 7 28,5
0,001 pg/μl std 8 31,4
blank 33,7

25. ALU 244
ALU 83
Cycle
Cycle
Std 3
Std 3 Std 4
Std 4 Std 5
Std 5
Std 6
Std 6 Std 7
Std 7
blank
blank
Threshold = 0,877
Threshold = 0,736
100 pg/μl std 3 13,5
10 pg/μl std 4 17,5
1 pg/μl std 5 21,3
0,1 pg/μl std 6 25,5
0,01 pg/μl std 7 28,7
blank neg
100 pg/μl std3 12,4
10 pg/μl std4 17
1 pg/μl std5 21,3
0,1 pg/μl std6 25,3
0,01 pg/μl std7 29,1
blank neg

26. Nano Inspired Biomedicine LaboratoryNano Inspired Biomedicine Laboratory