An Integrated Capacitive Array Biosensor for the Selective and Real-Time Detection of Whole Bacterial Cells
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Presentation at the 4th International Symposium on Sensor Science (I3S 2015) - Basel (July 13-15, 2015).
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An Integrated Capacitive Array Biosensor for the Selective and Real-Time Detection of Whole Bacterial Cells
- 1. An Integrated Capacitive Array Biosensor for the Selective and Real-Time Detection of Whole Bacterial Cells Numa Couniot, Laurent A. Francis, D. Flandre laurent.francis@uclouvain.be 4th International Symposium on Sensor Science July 13-15, 2015, Basel
- 2. Problem definition and Challenges Matrix Detec+on levels In 27 nL Blood 1 bacteria/mL 1 bacterium (p = 0.003%) Breast milk 103 bacteria/mL 1 bacterium (p = 3%) Urine samples 105 bacteria/mL 3 bacteria • How can we detect such a little amount of bacteria? For a 300 µm x 300 µm sensor in a 300 µm-thick channel • Selectivity? • How to deal with different solutions? 1 Staphylococcus aureus/mL must be detected among: • 109 red blood cells/mL • Possibly other non-pathogen bacteria Known problem for electronic biosensors (FET, impedance, etc.): à the screening from the surface properties (e.g. electrical double layer) at high salinity
- 3. Problem definition and Challenges Electrode Double Layer (DL) ~ 1-30 nm Bacterial cell ~ 1 µm Electrolyte • How to deal with different type of solutions/electrolytes? Electrical potential --------- + + + + + + + + + + + + + -------- --- +++ Strongly depend on the ionic strength!!!
- 4. Problem definition and Challenges Electrode Double Layer (DL) ~ 1-30 nm Bacterial cell ~ 1 µm Electrolyte Surface effects à Low sensitivity Volume effects à High sensitivity Go to High Frequency! 1 bact. = ~70 aF • How to deal with different type of solutions/electrolytes? Strongly depend on the ionic strength!!!
- 5. 1 µm Bacteria Transducer Selective agent Readout interface Staphylococcus epidermidis à Similar to S. aureus à Non-pathogenic Interdigitated microelectrodes à High active area à Similar size as bacteria à Electric field in surface Lysostaphin à Selectively destroys bacterial cell wall à Extendable to most bacteria with lysins CMOS à Low-cost à Miniaturization à System integration BIOLOGY SENSOR BIO/CHEM. ELECTRONICS CMOS capacitive array biosensor Our approach
- 6. Electrolyte capacitance monitoring Interdigitated microelectrodes (IDE) Integrated pixels Integrated oscillator Electrokinetic effects Four steps to get to bacterium detection 1 2 3 4
- 8. Interdigitated microelectrodes (IDE) [Couniot et al., Biosensors and Bioelectronics, 67, pp. 154-161, 2015] TOP VIEW CROSS SECTION +50 mV -50 mV +I0 -I0 +50 mV -50 mV Cins Rsol Cins Csol ALD-alumina passivated electrodes
- 9. Time [min] Y/ω[pF]#Bacteria[#/mm2] Optical Electrical [Couniot et al., Biosensors and Bioelectronics, 67, pp. 154-161, 2015] Real-time monitoring of S. epidermidis
- 10. No background noise Low-cost Robust to wash Advantages: Selectivity based on lytic enzymes (lysostaphin) [Couniot et al., Biosensors and Bioelectronics, 67, pp. 154-161, 2015]
- 11. Destroyed S. epidermidis causes decrease in capacitance Selectivity means based on lytic enzymes Urine with S. epidermidis (target) and E. faecium (control -) [Couniot et al., Biosensors and Bioelectronics, 67, pp. 154-161, 2015] 1. Naked 2. Ef 3. Ef + Se 4. Ef + Se(killed)
- 12. Same number of E. faecium, no impedance shift [Couniot et al., Biosensors and Bioelectronics, 67, pp. 154-161, 2015] Selectivity means based on lytic enzymes Urine with E. faecium only (control -) 1. Naked 2. Ef 3. Ef(not killed)
- 13. Selectivity means based on lytic enzymes Reproducibility of the normalized shift
- 15. ~ 1% of bacteria captured ~ 99% of bacteria lost ~ 1% of bacteria captured ~ 99% of bacteria captured Trap bacterial cells with electrokinetics How to decrease the LoD? FLOW FLOW
- 16. Macroelectrode For electrokinetic actuation Capacitive Sensor +7 V - 7 V 50 mV à Generate electrokinetics (EK) effects Design of the device [Couniot et al., Lab on chip, submitted, 2015]
- 17. LoD : 3.5.105 CFU/mL in 20 min à 11x better than without EK AC-Electroosmosis @ 10 kHz [Couniot et al., Lab on chip, in Press, 2015] Bacterial incubation 0 20 40 60 80 3.15 3.2 3.25 3.3 3.35 Time [min]||Y/ω||[pF] w/ AC-EO w/o AC-EO 7.10 CFU/mL 6 1.6 . 10 CFU/mL 7 ~5fF/min ~ 1 fF/min PBS 1:1000 PBS 1:1000
- 18. LoD : 105 CFU/mL in 20 min à 38x better than without EK OFF/ON Steps 0 20 40 60 80 3.15 3.2 3.25 3.3 3.35 3.4 3.45 3.5 3.55 Time [min]||Y/ω||[pF] w/ DEP & ET w/o DEP & ET Bacterial incubationPBS 1:1000 PBS 1:1000 7.10 CFU/mL 6 1.6 . 10 CFU/mL 7 Δ1 Δ2 Δ3 Δ4 Δ5 [Couniot et al., Lab on chip, in Press, 2015] Electrothermal + Dielectrophoresis @ 63 MHz à Flow-based method to direct bacteria from edge to the sensor center
- 20. VHF Capacitance-to-Frequency converter [Couniot et al., IEEE TCAS II, vol. 62, 2, 2015] M5 VHF GND M4 M3 M2 M1 CMOS Al2O3 DL Electrolyte VIA45 VIA Bacteria VHF CROSS SECTION Sensing Part Electronics
- 21. VHF Capacitance-to-Frequency converter [Couniot et al., IEEE TCAS II, vol. 62, 2, 2015] TOP VIEW
- 22. VHF Capacitance-to-Frequency converter en Vdd Vdd Vdd Five-stage ring oscillator Ten-stag Sub-interdigitated microelectrode arrays (M5) fIDE Five-stage ring oscillator 5 sub-interdigitated electrodes (M5) [Couniot et al., IEEE TCAS II, vol. 62, 2, 2015] Frequency divider ~ 300 MHz ÷ 1024 ~ 300 kHz OUTPUT 0 0 0 1 1 1 0 1 0 1
- 23. Intrinsic and extrinsic capacitances A factor 2 of difference despite the same coverage… f200 µm −1 ∝ 1pF + 0.77*Csol,200 µm( ) f100 µm −1 ∝ 0.8pF + 0.77*Csol,100 µm( ) 3.5 ± 0.1 pF 1 ± 0.025 pF The capacitance decreases à Assessed by simulations/models since cytoplasm dominates @ VHF à εr,cyto ≈ 70 < εr,PBS = 80 Before After Bacterial sensing in pure PBS 10 6 0 Sensitivity[%] 200 μm-sided: exp. #1 exp. #2 mean 100 μm-sided: exp. #1 exp. #2 mean 4 8 12 10 7 10 8 10 9 fIDE [Hz] ~ 150 MHz [Couniot et al., IEEE TCAS II, vol. 62, 2, 2015]
- 25. Single bacterial cell Reduce the sensor size Bacterial binding Δ1 Nominal sensor capacitance: 100% Single bacterial cell Δ2 [Couniot et al., IEEE TBCAS, in Press, 2015] How to improve sensitivity & multiplexing?
- 26. Single bacterial cell Problem: the bacterial cell can be outside the sensor Bacterial binding Δ1 Nominal sensor capacitance: 100% How to improve sensitivity & multiplexing? Single bacterial cell Δ2=0 [Couniot et al., IEEE TBCAS, in Press, 2015]
- 27. Single bacterial cell Solution: make a sensor array Bacterial binding Δ1 Nominal sensor capacitance: 100% Single bacterial cell Δ2 [Couniot et al., IEEE TBCAS, in Press, 2015] How to improve sensitivity & multiplexing?
- 28. System architecture (Top view) [Couniot et al., IEEE TBCAS, in Press, 2015]
- 29. System architecture (correlated double sampling) [Couniot et al., IEEE TBCAS, in Press, 2015] Vdd Vdd Vdd MR MBUF MSELCIDE CD MC1 MC2 MINIT Vc1 Vc2 MT MSHR Vshr CSHR MSHS Vshs CSHS MB Vb Vsel Vr Pixel (i,j) Column Amplifier (j) Columnbus Vinit VIDEVref Vpix VgT Buffer + Row select. Charge sharing principle Subthresh. Gain Csol Cins σsol Ideal linearity −50 0 50 0.12 0.16 0.2 0.24 0.28 Variation of parameters [%] VgT [V] σsol = 1.8 mS/m Csol = 55.6 fF Cins = 500 fF Rsol = 7 MΩ CDL = 4.5 pF Csol Rsol Cins CDL Cins CDL CIDE PIXEL TOP VIEW
- 30. 0 10 20 30 40 50 −10 −5 0 5 Time [min] Vout [mV] 1 3 4 PBS 1:1000 w/ bacteria PBS 1:1000 w/o bacteria 2 Pixel (2,7) Pixel (2,6) Pixel (2,8) w/o bacteria w/ bacteria Real-time monitoring [Couniot et al., IEEE TBCAS, in Press, 2015] w/o bacteria w/ bacteria Type 1 Type 2 w/o bacteria w/ bacteria Type 1 Type 2 *
- 31. w/o bacteria w/ bacteria [Couniot et al., IEEE TBCAS, in Press, 2015] Real-time monitoring *
- 32. [Couniot et al., IEEE TBCAS, in Press, 2015] Real-time monitoring 0 Number of bacteria Simulation 2 4 6 8 10 12 0 10 20 30 0 0.23 0.46 0.69 ΔVout[mV] ΔCsol[fF] Number of bacteria 0 -20 Experimental #5 ΔVout[mV] 5 10 15 20 0 20 40 #6 #7 #11 #11 #23 #13 #20 #12 #9 #10 #3 #8 #8 VIDE Vgnd VIDE Vgnd ≠ ΔC ≈ 167 aF ΔC ≈ 38 aF [Couniot et al., Sensors and Actuators B, vol. 189, pp. 43-51, 2013] VIDE Vgnd ≠ ΔC ≈ 7 aF
- 33. CMOS capacitive array biosensor Interdigitated microelectrodes (IDE) Integrated pixels Integrated oscillator Electrokinetic effects 1 2 3 4 High sensitivity @ high salinity High sensitivity By bacterial trapping Single Bacteria Detection Selectivity
- 34. F.R.S.-‐FNRS Funds D. Bol, J. Mahillon & J-‐L. Gala for their supervision T. Vanzieleghem & J. Mahillon for their biological exper+se J. Rasson & N. Van-‐Overstraeten benefits for useful discussions O. Poncelet for ALD deposi+on C.A. Dutu for technical help with PDMS cap micro-‐fabrica+on D. Spôte for the fabrica+on of the pressure tool UCL WINFAB plaoorm for help with micro-‐fabrica+on UCL WELCOME plaoorm for help with measurement setup Thank you for your attention! Questions? laurent.francis@uclouvain.be Acknowledgements