A CMOS Label- -free DNA free DNA A CMOS Label Microarray - PowerPoint PPT Presentation

A CMOS Label- -free DNA free DNA A CMOS Label Microarray Microarray Erik Anderson Stanford University I2MTC 2008

Motivation Motivation • Affymetrix + Agilent alone had $2.4 billion (USD) in revenue in 2007 for bio-analytic measurements • Drug discovery • Diagnostics • Research • Forensic testing • Growing interest in personalized medicine • Therapeutics tailored to your genetic profile • Conventional microarrays are expensive, big bulky systems (optics, lasers, reagents) • Can we leverage integrated circuit fabrication techniques for a low-cost approach? www.dnavision.be 2 Erik Anderson

Outline Outline • Motivation • Background • Charge sensing of DNA polymerization • CMOS sensor • Conclusions 3 Erik Anderson

DNA DNA • Contains genetic instructions to construct and regulate cellular components • Consists of 4 nucleotides • Adenine (A), Thymine (T), Cytosine (C), Guanine (G) • Usually found double-stranded, but single-stranded version exists too • A only binds with T, C only binds with G 4 Erik Anderson

Microarray Basics I Basics I Microarray Fluorescent Label Target ssDNA Probe ssDNA www-als.lbl.gov A A A C C C G G G G T T A C G C A A A T T G C A A T A T C A G C G G C G G C G C G C G C C C G C T A C T A T A T C T A T A T G T A G Spot 1 Spot 3 Spot 2 5 Erik Anderson

Microarray Basics II Basics II Microarray Gene Chip Microarray Scanner – Image Cost: ~$200k Affymetrix Gene Chip • Light from a grid location indicates the presence of the corresponding target in a sample • Limitations: Expensive and not portable Images courtesy of Affymetrix 6 Erik Anderson

TAG4 Example from SGTC TAG4 Example from SGTC • TAG4 = yeast genome used with optical scanners • Run time – DNA Extraction 2 hr – PCR & labeling 2 hr – Hybridization preparation 0.5 hr – Hybridization 6-16 hr – Wash & Stain 3 hr – Scan of chip 0.25 hr • Cost per chip (“Academic Prices”) – Chip $150-300 – Reagents $50-150 100,000 features or “spots” which are 8 � m x 8 � m • • Probes are 20 nucleotides in length • Targets range from 100-200 nucleotides – 10-100 ng/mL amplified (PCR) to concentrations of 1 � g/mL • Works well when you are interested in massively parallel detection – Suitable for point-of-care applications? 7 Erik Anderson

Post Processing Challenges Post Processing Challenges Thewes et al. ISSCC 2002. Han et al. ISSCC 2007. 8 Erik Anderson

System Requirements System Requirements • Suitable for point-of-care applications – Leverage IC fab technology for low-cost approach – Label-free – Easy post-processing – Integrate microarray with the “readout” – Reduced number of features from conventional optical techniques – goal is 25 • Detects targets at 10 � g/mL 9 Erik Anderson

DNA Polymerization DNA Polymerization Targets Polymerase Probes A A C C G G A T T C A C A T T C A T C A T G G G C G C C C G C C G T T A T T A T T T A T A Second strand CANNOT Second strand CAN be synthesized be synthesized Polymerase works at double-strand / single-strand junctions 10 Erik Anderson

Principle of Detection Principle of Detection dNTP (e.g. A, T, C, G) T A Polymerase A T G C A T e - H + G C G C C A Electrode + Transient current • System detects a NON-equilibrium charge distribution 11 Erik Anderson

Polymerization Chemical Reaction Polymerization Chemical Reaction 3’ 5’ O O P O P O O Double-stranded Primer O O + DNA DNA H Mg Probe DNA 4. Liberated H + , PPi·Mg and catalytic O Base Base O Mg 2+ 3’ O 3. New fixed H - O O O charge P O O O P O P O O P O O Base Base Base O O O O Mg : O : O H H Not drawn to scale 1. Enzyme + dNTP·Mg Base 2. Nucleotide + catalytic Mg 2+ incorporation SAM + 5. Induced charge 12 Erik Anderson

Induced Charge Induced Charge What fraction of a charge is induced on a nearby electrode? Electrode location Immobilize DNA close to electrode Charge is 0.1 electrode- to maximize induced charge widths above electrode 13 Erik Anderson

CMOS System Requirements CMOS System Requirements • Linear, monotonic signal response • “Low power” ( back-of-envelope estimate, ≤ 42 mW) – Die surface temperature should not rise more than 1 ° C above ambient over 5 minutes • “Low noise” – Amplifier noise ≤ other system noise contributions • Electrode area large enough for spotting DNA onto electrodes ( ≥ 100 – 200 square � m) • Easy post-processing • ± 1 V swing at output (use thick gate-oxide devices) 14 Erik Anderson

CMOS Architecture CMOS Architecture 15 Erik Anderson

OTA OTA V hi V cm V b V b In+ In- V o V b Common-mode feedback V lo Input V o,hi In+ In- V b V b V b V b V o,lo In+ In- Bias Bias 16 Erik Anderson

OTA Specs OTA Specs 0.18 � m CMOS (3.3V devices) Technology Gain 110 dB Gain(Vo = 1V) 63 dB Gain(Vo = -1V) 82 dB Phase Margin 75° CMRR 110 dB PSRR+ 70 dB PSRR- 110 dB Unity Gain 250 kHz Power per pixel 1.7 mW Simulated for typical corner at 75 ° C 17 Erik Anderson

Reset Logic Reset Logic Saturation Detector - + Used to extend dynamic range 18 Erik Anderson

Easy Post- -Processing Processing Easy Post Polymer that we apply Standard Passivation from fab CMOS fab Electrode in top metal Silicon 19 Erik Anderson

Temporal Noise Temporal Noise • Noise spectral density is not the right analysis • Signal is observed in time → want time domain noise • Temporal noise = variance of noise at a particular instant in time = Temporal noise 20 Erik Anderson

Electronic Noise Contributions Electronic Noise Contributions Equation Value @1 sec Comments 244 � V ω Flicker max 2 K ln( ) − 10 2 f max = 250 kHz K f = 2 . 4 × 10 V f min = 1 Hz f ω min 19.5 � V Thermal 2 nV A ω V 2 = GBW = 250 kHz o o n V n 22 2 Hz Voltage 10.5 � V Thermal 2 I n t fA C 1 = 30 pF 2 = int 1 I n 2 C Current Hz 1 11.7 � V kT Cap. Reset T = 300 K C 1 = 30 pF C 1 6.0 � V 2 qI avg t Shot int I avg = 1 pA C 1 = 30 pF 2 C 1 21 Erik Anderson

Die Photo + Test Board Die Photo + Test Board Pixel 300 � m Die Bondwires encapsulated in epoxy 22 Erik Anderson

Minimum Detectable Current Minimum Detectable Current • Enzymatic buffer noise is constant w.r.t. integration time ~830 � V RMS • Limit of detection with buffer is 25 fC • Corresponds to biological limit of detection of 8 ng/mL (worst case) • Crosstalk dominated by system noise → not measurable Noise from enzymatic buffer dominates electronic noise 23 Erik Anderson

Measured Signal from CMOS Chip Measured Signal from CMOS Chip Probe: GTG CCA AGT ACA TAT GAC CCT ACT CAC GGT TCA TGT ATA CTG GGA TGA CCA TAC CTG TAC GAC TCG AGT GAC GAG ACG GCG TA Exposed segment • Target concentration 10 � g/mL 24 Erik Anderson

Conclusions Conclusions • Designed first CMOS DNA polymerization sensor – Targeted to low-cost, point-of-care applications – Demonstrated sensor could detect useful concentrations 25 Erik Anderson

Supplemental Supplemental • Following slides are supplemental 26 Erik Anderson

Future Applications Future Applications • Clinical, point-of-care diagnostics • Personalized medicine – Enabled by low cost fab techniques • Pathogen detection • Short segment DNA sequencing – Sequentially add nucleotides and observe the signal • Simple Nucleotide Polymorphism (SNP) Detection – SNP = an alteration in a few nucleotides, e.g. AAAA vs. ATAA – SNPs form 99.77% of all genetic variation 27 Erik Anderson

Pathogen Detection Pathogen Detection Add A, T, G, C Polymerase A A C C G G A T T C A A C T T C A T C A T G C G G G C C C G C C G T A T T T A T T A T A T Spot 1 Spot 2 28 Erik Anderson

SNP Detection SNP Detection Add A, T, G, C Polymerase A A C C G G A T T A A A A T T A T A T G G G C G C G G C G C G C C T A T A T T T A T A T T Spot 1 Spot 2 • Trick is to make the mutation part of the target • Add nucleotides and read out a signal at spots where SNP is present 29 Erik Anderson

Short Segment Sequencing Short Segment Sequencing A T C A A A A C C C C G G C G C G C G C G A A T T C G A C G A T T G G A T C C A T G C G C G C G C C G C G C G C G T A T A T A T A T A T A T A T A Spot 1 Spot 2 • Sequentially add bases • Wash away unused bases between additions 30 Erik Anderson