Microchip-based Sensors for Detection of Magnetic Microbead Labels
Transcription
Microchip-based Sensors for Detection of Magnetic Microbead Labels
Microchip-based Sensors for Detection of Magnetic Microbead Labels Mark Tondra Diagnostic Biosensors, LLC; 1712 Brook Ave. SE; Minneapolis, MN 55414 [email protected] 612 331-3584 www.diagnosticbiosensors.com Oak Ridge AACC 4-20-06 MagSensors Outline • Using magnetic beads as assay labels • Magnetic sensor chips for tiny and disposable integrated assay systems • Micromagnetic detection platforms – Flowing magnetic labels / towards cell counting – Immobilized labels on the sensor chip – Immobilized labels on a separate chip + scan • Conclusions Oak Ridge AACC 4-20-06 MagSensors Acknowledgements •Funded by NSF, DARPA CAMD ISU Dept. of Chemistry: Dr. Marc Porter John Nordling Rachel Millen Nikola Pekas Toshi Kawaguchi NVE GMR sensor fab: LSU – CAMD Dexin Wang microfluidics: Zhenghong Qian Jost Goettert Anthony Popple Changgeng Liu Dave Brownell Zhengchung Peng Bob Schneider Kun Lian Kevin Jones Josef Hormez Loren Hudson Challa Kumar John Taylor Albrecht Jander Oak Ridge AACC 4-20-06 MagSensors NRL assay development: Lloyd Whitman Jack Rife Cy Tamanaha Mike Miller Shaun Mulvaney Making Biology Magnetic • Attach magnetic particles to bio-species – Sizes range from 10 nm to 5 µm diameter – Commonly ~10% magnetic content • Trade-offs – More magnetism may cause aggregation – Larger beads give bigger signal, but dominate the dynamic properties of the analytes in solution • Unique chemical binding for each species – Special surface coatings and treatments Oak Ridge AACC 4-20-06 MagSensors Benefits of using Magnetic Labels for Biosensors • Very low magnetic background • Single label detection • Magnetic forces can enhance hybridization rates • Magnetic forces can reduce non-specific binding • Wide range of sizes and magnetic properties available Oak Ridge AACC 4-20-06 MagSensors Immobilized analytes: “two-probe” DNA assay (NRL) C) Introduce magnetic labels with unique “label probes” D) Magnetic labels bind only where they match B) Introduce fluid sample carrying potential molecular match, allow to hybridize M M M E) Read magnetic detector array by applying a magnetic field, measuring the resistance change Spintronic Detector Spintronic Detector A) Prepare array chip by spotting DNA “capture probes” onto surface Oak Ridge AACC 4-20-06 MagSensors Stray Fields from Bound Magnetic Nanolabel Detector sees HTotal = Happlied + Hstray along a designed sense-axis Happlied M Hstray Spintronic Detector Oak Ridge AACC 4-20-06 MagSensors Motivation for Recent R&D Efforts Military / Homeland Defense wants bioassays that are: •Rugged •Lightweight / handheld •Cheap •Rapid •Highly sensitive and specific, multi-functional, foolproof •Readers, sensors, and fluidics must be massmanufacturable. Oak Ridge AACC 4-20-06 MagSensors Laboratory-on-a-Chip •Shrink clinical or diagnostic laboratory setup onto a Sitype chip •Point-of-Care, Point-of-Use applications •Uses MEMS and microfluidics •Make technology available to: soldiers in field, security workers, eventually consumers •Not yet a commercial reality Oak Ridge AACC 4-20-06 MagSensors Giant MagnetoResistive (GMR) design: Microfluidic Channel over Detector Two detectors are shown here. One is downstream from the other by 100 microns. Each detector has two reference and two sensing GMRs configured as a Wheatstone bridge E1 RR1 GND Isrc RS1 RS2 RR2 Top View E2 Channel passes over sensing GMRs Oak Ridge AACC 4-20-06 MagSensors Voltage / Resistance Idealized Linear GMR Detector Resistance vs. Magnetic Field Resistance ∝ R0 + sinθ θ Sense Layer Pinned Layer -20 -10 0 10 20 Field (Oe) A GMR thin film resistor is typically a micron wide, 0.01 micron thick, and arbitrarily long. Oak Ridge AACC 4-20-06 MagSensors Voltage / Resistance Resistance changes when magnetic labels are present Resistance ∝ R0 + sinθ Signal is resistance difference θ Pinned Layer Resistance with labels -20 -10 0 10 Field (Oe) Oak Ridge AACC 4-20-06 MagSensors Sense Layer 20 Array of 20 GMR biosensors under serpentine microfluidic channel ~150 µm wide fluidic channel ~200 µm diameter sensor spot (matches pin spotter size) Oak Ridge AACC 4-20-06 MagSensors Micromagnetic Detection Platforms 1. Detect magnetized objects in a microfluidic flowstream (e.g. cytometer) 2. Detect magnetic beads bound to the sensor surface (may include microfluidics) 3. Detect magnetic beads bound to a different surface (e.g. glass slide) Oak Ridge AACC 4-20-06 MagSensors Detection of Flowing Magnetics • Towards a cell counter or “cytometer” Oak Ridge AACC 4-20-06 MagSensors Flow Cytometer working definition •Counts cells in a continuously flowing system •Usually needs to discriminate between various cell types (e.g. all healthy cells vs. cancerous cells) Oak Ridge AACC 4-20-06 MagSensors Magnetic Flow Cytometer Phases 1. ISOLATE cells of interest magnetic labels 2. IDENTIFY them magnetic labels 3. DIRECT them to detector magnetic / fluidic forces 4. COUNT the cells in flow Giant Magnetoresistive (GMR) detector Oak Ridge AACC 4-20-06 MagSensors 1. ISOLATE Cells of interest 1. ISOLATE cells of interest magnetic labels 2. IDENTIFY them magnetic labels 3. DIRECT them to detector magnetic / fluidic forces 4. COUNT the cells in flow Giant Magnetoresistive (GMR) detector Oak Ridge AACC 4-20-06 MagSensors Prototypical Example: Cancer Cell Isolation •Want to grab only cancerous cells, count them, and store them for further analysis •Could be only 1 : 109 •May not be distinguishable by size or color Oak Ridge AACC 4-20-06 MagSensors Magnetic isolation is a common approach •Add special magnetic particles to container Oak Ridge AACC 4-20-06 MagSensors Biochemically specific attachment •Add special magnetic particles to container •Allow specific binding of labels to cell Oak Ridge AACC 4-20-06 MagSensors Apply magnetic force •Add special magnetic particles to container •Allow specific binding of labels to cell •Use magnet to attract cells to corner Oak Ridge AACC 4-20-06 MagSensors Remove “chaff” from system •Add special magnetic particles to container •Allow specific binding of labels to cell •Use magnet to attract cells to corner •Dump out waste Oak Ridge AACC 4-20-06 MagSensors 2. Give cells of interest a unique IDENTITY 1. ISOLATE cells of interest magnetic labels 2. IDENTIFY them magnetic labels 3. DIRECT them to detector magnetic / fluidic forces 4. COUNT the cells in flow Giant Magnetoresistive (GMR) detector Oak Ridge AACC 4-20-06 MagSensors Cell is IDENTIFIED with same labels •Add special magnetic particles to container •Allow specific binding of labels to cell •Use magnet to attract cells to corner •Dump out waste •Add water •Repeat as needed Oak Ridge AACC 4-20-06 MagSensors 3. DIRECT Cells to a Detector 1. ISOLATE cells of interest magnetic labels 2. IDENTIFY them magnetic labels 3. DIRECT them to detector magnetic / fluidic forces 4. COUNT the cells in flow Giant Magnetoresistive (GMR) detector Oak Ridge AACC 4-20-06 MagSensors Single-Wire Magnetic Director Top view X-sections Channel: 400 µm long Hext Electrical current Flow 50 µm wide channel Simple situation: Qualitative force calculation Hx = Hexternal = 100 Oe 35 µm deep 50 µm wide channel x X-section 1 µm diameter Paramagnetic Small wire x-section Hexternal across channel Hexternal parallel Hwire Current into plane 1 µm x 1 µm wire under channel center Simple situation: Qualitative force calculation Hx = Hexternal = 100 Oe 50 µm wide channel Particles are attracted (Flip sign of current, particles are repulsed) 35 µm deep 1 µm diam. Particles paramagnetic Small wire x-section Hexternal across channel Hexternal parallel Hwire x Current into plane 1 µm x 1 µm wire under channel center Magneto-fluidic Dynamics For a given Fmag, one can calculate: Equation of motion dv m = − 3π η av + Fmag dt Viscous drag Magnetic force Integrate to get velocity 3π η a − t Fmag 1− e m v(t ) = 3π η a “terminal velocity” “characteristic time” a: particle diameter = 1 micron n: viscosity (water) m: particle mass Fmag: Force in channel cross-section due to Hwire and Hexternal v: velocity t: time Oak Ridge AACC 4-20-06 MagSensors Initial motion of particle far from wire Hx = Hexternal = 100 Oe 35 µm deep 50 µm wide channel Flow into page x Initial Fmag ~ 9 picoN Initial Vterminal ~ 1100 µm/sec Characteristic time = 87 nsec Max travel time = 0.03 sec. Because the characteristic time is so much smaller than the total travel time of the particle [overdamped], one can basically say that the particle trajectory follows the Current = 10 mAmagnetic lines of force Two-way Diverter Design and Results Top view X-section • A uniform external field magnetizes particles • Current lines induce field gradients of 102-103 T/m • Resulting force diverts particles to a desired channel Oak Ridge AACC 4-20-06 MagSensors Magnetic Flow Sorting Demonstration Top Views Bangs Labs, 28% magnetite, 1 µm Flow rate: 6 nL/min 85% of the beads in desired channel Oak Ridge AACC 4-20-06 MagSensors 4. COUNT the Cells in Flow 1. ISOLATE cells of interest magnetic labels 2. IDENTIFY them magnetic labels 3. DIRECT them to detector magnetic / fluidic forces 4. COUNT the cells in flow Giant Magnetoresistive (GMR) detector Oak Ridge AACC 4-20-06 MagSensors Detection of magnetic objects in flow •Giant Magnetoresistive (GMR) detector •Microfluidic flow channel passes directly over GMR detector Oak Ridge AACC 4-20-06 MagSensors Proof of principle: GMR Sensing of Magnetic Picodroplets Picoliter-sized droplets of ferrofluid formed at a fluidic junction Plug dimensions: 13 µm wide 18 µm deep 85 µm long FerroTec 307 10nm ferrite particles ~1% by volume GMR sensitivity 0.07%/Oe Pekas et al., Appl. Phys. Lett., 85, (2004) • Wheatstone bridge configuration T. Thorsen, R. W. Roberts, F. H. Arnold, and S. R. Quake, Phys. Rev. Lett., 86, 4163 (2001) H. Song, J. D. Tice, and R. F. Ismagilov, Angew. Chem. Int. Ed. 42 (7), 768 (2003) Oak Ridge AACC 4-20-06 MagSensors Direct Flow Velocity Monitoring flow Excitation field 15 Oe; Flow rate 250 nL/min; 1.2% magnetite v/v Velocity determined by cross-correlating the signals from two bridges Oak Ridge AACC 4-20-06 MagSensors Detection of single ~5 micron beads in flow Top View Model data from cell covered by “shell” of magnetic labels Hypothetical cell covered with magnetic labels Hx component, 2 µm below the 200 nm shell FEMLAB package Protozoan cell 8x6x6 µm 48 1-µm spheres, χ=0.3 (Dynal MyOneTM) Homogeneous 1-µm shell, χ=0.18 Homogeneous 200-nm shell, χ=0.2 (Micromod nanomag-D, χ=2) Detection of labels and cells in small channels is magnetically easy But, channel gets plugged new design 35 µm deep 50 µm wide channel old design 12 µm x 15 µm channel x-section 2 µm x 2 µm detector area GMR detector 3 µm x 15 µm detector area Design of Cell – Label splitter Top View Hext Redirect Split Discriminate Electrical current Gather Flow Cell sorter, director, and detector Oak Ridge AACC 4-20-06 MagSensors Fluid dynamics provide additional tools for microfluidic control This talk has largely ignored the fluid dynamics. However, they are very important! Mostly, a detailed account would show that there are additional tools that can be designed in to aid in sorting and detecting. Oak Ridge AACC 4-20-06 MagSensors New low profile fabrication process design a) Motivation: 1. 2. 3. 4. Need wider channels to avoid plugging Lower surface topography for better flow and sealing Want thin cover for closest microscope working distance Improved manufacturability b) Features 1. 2. 3. 4. 5. 6. Buried interconnects formed using damascene process Allows arbitrary channel width and alignment Much lower surface step height (<100 nm vs. 2000 nm) Thin passivation is viable (<100 nm) Facilitates electrodes for electrochemistry and applying electric forces Fluidic through-holes for better optical access and fluidics options Oak Ridge AACC 4-20-06 MagSensors Detecting Magnetic Labels bound to the Biosensor Surface 1. Multi-sensor array is convenient 2. Dynamic range of better than 3 logs (example: one detector can quantify from 5 to 5,000 labels on a 200 micron diam. Spot) 3. Low cost microchip is disposable 4. Requires microfluidics integration Oak Ridge AACC 4-20-06 MagSensors Array of GMR sensors under serpentine microfluidic channel ~150 µm wide fluidic channel ~200 µm diameter sensor spot Oak Ridge AACC 4-20-06 MagSensors NRL “cBASS” system Oak Ridge AACC 4-20-06 MagSensors NRL “cBASS” system, Array of GMR sensors 2.8 micron DynalBeads 1) 2) 3) 4) Courtesy L. Whitman, Naval Research Lab. Single label detection is possible >3 decades of dynamic range Better than 1 fMolar with fluidics Magnetism enhances specificity Oak Ridge AACC 4-20-06 MagSensors NRL “cBASS” system cBASS™ Prototype compact Bead Array Sensing System Oak Ridge AACC 4-20-06 MagSensors Detecting Magnetic Labels bound to a separate surface 1. Multi-sensor array is convenient 2. “Scan” the assay slide by the reader chip 3. Dynamic range of better than 3 logs Detector is “permanent” in reader 4. Much simpler sample handling technology 5. Increased mechanical engineering challenges Oak Ridge AACC 4-20-06 MagSensors Sample Strip Reader • Developed a GMR test station capable of detecting samples which are scanned across a sensor (e.g., magnetic card reader) • Developed a method for normalizing the GMR signal from samples at varying separations • Investigating analytical figures of merit • Potential utilization of streptavidin magnetic particles as a universal label Oak Ridge AACC 4-20-06 MagSensors GMR Test Station Electromagnetic Power Supply GMR John Nolding Digital VoltMeter Current Source Sample Break out Box Toshikazu Kawaguchi Single GMRs Coil Coil RR1 & RR2 Sample Above GMR GMR and PC connector SAMPLE MOVEMENT Test Station: power supply, voltmeter, and current source RS1 & RS2 400 µm All controlled by software written in house Oak Ridge AACC 4-20-06 MagSensors Sample Stick Experiment Move sample across GMR sensing area 200 x 200 μm SAMPLE MOVEMENT External field at 150 Oe Sample is held near the GMR (~50 µm) and moved across sensing area Permalloy Gold 500 μm Oak Ridge AACC 4-20-06 MagSensors Effect of Sample Leveling A N 1 mm 225 225 224 224 223 223 Signal (mV) Signal (mV) Internally referenced sample: 20-nm thick permalloy squares Signal After Sample Leveling Signal Before Sample Leveling 222 221 220 N A 219 222 221 220 219 218 A N 218 60 80 100 120 140 160 180 200 220 80 100 Time (s) 120 140 160 180 Time (s) Peak ID Signal (mV) Std. Deviation Peak ID Signal (mV) Std. Deviation A 4.91 0.07 A 5.02 0.03 N 3.72 0.07 N 4.97 0.05 Difference: 1.2 ± 0.1 mV n=8 Difference: Oak Ridge AACC 4-20-06 MagSensors 0.05 ± 0.06 mV 1 mm Raw Signal at varied Sample/GMR Separation 3.0 Signal (mV) 2.5 2.0 1.5 "0" µ m +50 µ m +100 µ m +150 µ m +200 µ m 1.0 0.5 0.0 200 400 600 800 Normalized Signal (signalx/signal800 µ m3) Multiple Sample Signal Normalization Normalized Signal at varied Sample/GMR Separation 1.2 0µm 50 µ m 100 µ m 150 µ m 200 µ m 1.0 0.8 0.6 y = 1.34 × 10 − 3 x + .0192 r 2 = 0.996 0.4 0.2 0.0 200 Sample Volume (µ m3) Oak Ridge AACC 4-20-06 MagSensors 400 600 Sample Volume (µ m3) 800 Permalloy Limit of Detection Measurements Sample 1 mm Parameters • Arrays of 24 x 24 µm squares • 20 nm thick permalloy • 12.2 µm3 permalloy which translates to ~31 magnetic particles (40% permalloy) on a 200 x 200 µm sensor Raw signal for permalloy sample 0.8 Raw Signal (mV) y = 1.97 × 10 − 3 x + .014 0.6 Predicted Limit of Detection (LOD): R 2 = 0.972 • 0.1 1µm MP/µm2 sensor detectable 0.4 Ways to Improve: 0.2 • Smaller GMR sensor 0.0 • Decrease GMR/Sample Separation 0 50 100 150 200 250 300 • 3) Sample volume (µ m Oak Ridge AACC 4-20-06 MagSensors Faster signal acquisition (signal averaging) MP Binding to Sample Stick Gold Square Permalloy Square *10 μL of MP on the patterned sample overnight Results: Binding to biotinylated gold square is preferred Non-specific binding to permalloy and pyrex is an issue Oak Ridge AACC 4-20-06 MagSensors Signal w/ Referenced Sample Stick A B C D E F G H I J K L M Sample Permalloy Gold Magnetic field: 150 Oe GMR-sample stick separation: ≤ 50 μm Oak Ridge AACC 4-20-06 MagSensors Signal (mV) # of MP A 0.141 1766 B 0.061 608 C 0.032 6 D 0.008 5 E 0.040 0 F 0.007 0 G 0.038 0 H 0.013 0 I 0.049 0 J 0.156 2840 K 0.115 601 L 0.352 8661 M 0.268 1326 GMR Signal vs. MP Concentration Limit of detection (3x SNB) is 0.005 MP/μm2, or 215 MP per gold square. Oak Ridge AACC 4-20-06 MagSensors Magnetic Microchip Fabrication Cross Section Diagram SU-8 Lid Gold Bonding Pad M Channel Layer GMR Sensor M Encapsulated Channel M Fluid Port Silicon wafer Copper Interconnects and Strip Lines Oak Ridge AACC 4-20-06 MagSensors Magnetic Excitation and Data Collection Module •8 On-board signal preamps •Jumpers for sensor channels •Jumpers for coil driver Oak Ridge AACC 4-20-06 MagSensors Research and Development on Magnetic Biosensors around the globe • Nat. Labs and Universities – EU, Korea, China, Several US Labs • Commercial Efforts – Siemens, Seahawk Biosystems, MagneBiosensors, Magnesensors Oak Ridge AACC 4-20-06 MagSensors Other magnetic detection technologies • Hall effect • Anisotropic magnetoresistance (AMR) • SQUID • Coils Oak Ridge AACC 4-20-06 MagSensors Chip-based detection and manipulation advantages • Sensors and manipulators are on same length scale as labels, cells • Opens up many opportunities for very small systems: – Implanted diagnostic sensors – Catheters Oak Ridge AACC 4-20-06 MagSensors Conclusions • Magnetoresistive sensors are a versatile tool for biochemical research and development • Some sensors operate “wet,” some “dry” • Microfluidics and / or mechanics are next • Packaging and testing issues are current barriers to low-cost commercial disposable biosensor products • Laboratory standards are lacking Oak Ridge AACC 4-20-06 MagSensors Company Goal • Develop technology leading to commercially available diagnostic biosensors. Oak Ridge AACC 4-20-06 MagSensors