A handheld system for DNA test based on Lab on chip technologies. Marco Bianchessi

Transcription

1 A handheld system for DNA test based on Lab on chip technologies Marco Bianchessi

2 History 2 Mid 90s: Lab on chip introduction From niche to high-volumes products

3 What is needed: 3 Miniaturization euros euros 400 euros 120 euros 30 euros 5 euros Price of 1 Mbit of Memory 0,5 euro 0,05 euro Low cost 0,0001 euro Ease of use

4 Q3 system 4 Allows DNA/RNA tests (NAT) out of the molecular biology lab Instrument Disposable cartridge

5 A new system partition 5 LAB Instrument Disposable Low instrument cost: No need for huge initial investment Pay per use: No need to perform a minimum number of tests to be cost effective Point of care Simpler use: The disposable is preactivated and has the highest industry quality standards Basic trained personnel

6 Cost per test Initial investment Cost comparison Central Lab Point of Care PoC is cheaper Lab is cheaper Asymptotic Cost Number of tests 6

7 Ebola pandemic risk 7 Needs: Quickly identify ebola infected patients and separate them from others. Symptoms are easily confused with Malaria Control people at borders, to avoid the spreading of pandemic Available options Immunoassay based tests, but are low sensitivity and not very specific Send sample to central labs, but in Africa are very uncommon and the infected blood is very dangerous Advantages of NAT: NAT is now the gold standard for WHO, a new rapid test is straightforward

8 Meat-ID Food quality and safety 8 Given a sample of minced meat, it may contain unwanted traces of animals other than the declared one. The final manufacturer wants to ensure quality and avoid bad reputation if fraud is discovered. (in bona-fide) Today the internal quality-control labs of food industries does not perform genetic tests. Big companies may invest in that, small producers cannot afford it. Meat-IT tm measures contents of Beef, Pork, chicken and horse. It also detect a gene of high valuable piemontese beef Applications are: Avoid intentional/unwanted frauds Guarantee genuine piemontese beef Halal / kosher food certification (100% pork free)

9 How DNA can be detected? Realtime PCR is now the preferred method for most molecular biology applications. PCR is an enzymatic reaction that permits in vitro amplification of the DNA, to obtain identical copies of an initial molecule of DNA. It is possible to select a sequence of DNA, if it is present in the sample the reaction takes place, otherwise nothing happens. DNA amplifies exponentially and it becomes easily measurable. Realtime PCR can tell if a DNA sequence is present in the initial sample and can also quantify the amount.

10 What is needed to implement Realtime PCR? A way to precisely control the temperature. To amplify DNA several chemical steps must be done in the correct order Each step occurs only at a specific temperature. So: Heat is the handle that allows us to control what happens at nanoscale. 2. A way to precisely measure fluorescence (on multiple optical bands) 1. While DNA amplifies, it makes specific molecules (Dyes) to become fluorescent 2. Measuring fluorescence while the reaction happens allow us to probe what is happening to DNA 3. It is possible to associate different DNA to different Dye colors, increasing the information And a way to confine the reagents

11 Typical Realtime PCR system components 11 Peltier cooled alluminium plate Halogen lamp Moving filter carousel CCD sensor Polypropylene wells

12 Temperature cycles 12 Denaturation Extension Annealing Temperature Maintainment Temperature Change (ramps)

13 About speed: The maintaining times are defined by the PCR protocol: they depend on the reagents, the enzymes and the length of the target The ramps depend on the instrument: the faster the better. With standard (slow) enzymes, the total time spent for ramps is negligible, so there is not much gain in shortening it. With new enzymes, (fast) the ramp time is comparable with the maintaining time, so fast ramps shorten sensible the total test time. Combining fast enzymes and short ramps a typical test pass from 90 to 30 minutes. For point of care it matters

14 Importance of temperature precision T denaturation Too high: polymerase can be damaged Too low: DNA does not denature completely No reaction Low efficiency T hibridization Too low: primers my bind a-specifically, amplifying unwanted DNA Too high: Primers do not bind. Low efficiency / No reaction Wrong result T extension Too high: primers may detach during extension Too low: reaction slows down. Low efficiency Low efficiency / No reaction

15 Cartridge: the concept 15 Transparent plastic cover Label with barcode Top view Reagents under wax (self-sealing) Silicon chip + RFID Reaction chambers Heaters Bottom view (silicon chip) Temperature sensor Direct contacts on chip pads

16 Cartridge: surface treatment 16 A patterned hydrophobic layer for droplets confinement Mixture of PCR reagents Dry film Hydrophobic Silicon chip SiO 2 Hydrophilic

17 The chip is the thermal cycler 17 R1 (heater) R2 (Temp measure)

18 Closed loop temperature control 18 Chip Res. PID Controller Desired temperature Current Ω R T C

19 Calibration 19 Different chips may have different resistors values, due to process tolerances. For long and narrow lines it could reach few % R Ω T C Instead of considering R, that depend on geometric, consider ρ, that depends only on the material. Chip to chip variations depend only on impurities, it is negligible. ρ Ω T C

20 Chip calibration 2 20 Is correct to consider ρ constant for every chip. A precise and calibrated thermometer is placed in the instrument, to measure room temperature At equilibrium, the chip is at room temperature The resistance of the chip at known room temperature is measured, so a point of the R-T curve is known Knowing the slope ( related to ρ ) the whole curve is derived. The accuracy of the temperature measured in this way is less than 0.1 C

21 and temperature homogeneity 21 If there is a gradient in temperature, means that different wells will have different efficiency or inconsistent results. Silicon is a fairly good heat conductor, it minimizes thermal gradients over the surface. The heater resistor is made by several resistors in parallel (instead of being a single strip) If a point is hotter than the rest, its resistance increases. In a single strip resistor, the current is equal in every point, so the hotter point dissipates more energy, becoming even hotter (positive feedback) In a parallel bank of resistors, if a point is hotter it draws less current, dissipating less heat and becoming cooler (self balancing)

22 T C The faster the better! 90 Cooling Air fan Peltier cell 50 Peltier ΔT Room Temp. time 22

23 Thermal performances 23 Using closed loop control we reached a temperature resolution of 0.1 C Using on-site calibration we reached a thermal accuracy of 0.2 C Thanks to uniform heating resistor and silicon s good thermal conductivity, we reached a thermal uniformity of 0.2 C Thanks to silicon s thermal conductivity, low thermal inertia and the relative high area of dissipation, we reached a cooling rate higher than 4 C/s Thanks to silicon s thermal conductivity, low thermal inertia and local resistor thermally coupled, we reached a heating rate higher than 10 C/s Thanks to the specific architecture, these performances are really referring to the reaction volume, (not to the cooling plate)