Layer A: PDMS Microfluidics Testing and Protocol Optimization Testing and Protocol Optimization Steps Towards Mass Production Steps Towards Mass Production Sebastian J. Osterfeld Sebastian J. Osterfeld 1 1 , , Shu Shu - - Jen Han Jen Han 1 1 , Stefano Caramuta , Stefano Caramuta 2 2 , , Liang Liang Xu Xu 1 1 , , Heng Heng Yu Yu 2 2 , Jin Xie , Jin Xie 3 3 Shouheng Shouheng Sun Sun 3 3 , , Nader Nader Pourmand Pourmand 2 2 , Shan X. Wang , Shan X. Wang 1 1 1. Stanford University 1. Stanford University 2. Stanford Genome Technology Center 2. Stanford Genome Technology Center 3. Brown University 3. Brown University (Project funded by DARPA) (Project funded by DARPA) Testing and Fabrication of the MagArray Testing and Fabrication of the MagArray ® ® Biochip Biochip Our aim is to repeatedly test prototype chips with a variety of Our aim is to repeatedly test prototype chips with a variety of nanoparticles nanoparticles , biological molecules, and various , biological molecules, and various experimental protocols, to determine and improve the performance experimental protocols, to determine and improve the performance characteristics of the MagArray. For maximum characteristics of the MagArray. For maximum flexibility, each chip needs to be individually functionalized w flexibility, each chip needs to be individually functionalized w ith ith biomolecules biomolecules . As a result, the prototype chips employ . As a result, the prototype chips employ sensors on a freely accessible surface, and macroscopic fluidics sensors on a freely accessible surface, and macroscopic fluidics are employed instead of are employed instead of microfluidics microfluidics to deliver the to deliver the reagents during the measurement. reagents during the measurement. Left: A prototype MagArray4 chip, made Left: A prototype MagArray4 chip, made at Stanford. Inside the well are two freely at Stanford. Inside the well are two freely accessible arrays, each containing 16 accessible arrays, each containing 16 individually addressable sensors. At the individually addressable sensors. At the Stanford Genome Technology Center, the Stanford Genome Technology Center, the 16 sensors on the left are manually 16 sensors on the left are manually functionalized with a different functionalized with a different biochemistry than the 16 sensors on the biochemistry than the 16 sensors on the right. Additional sensors, in this case 32, right. Additional sensors, in this case 32, are covered with epoxy and serve as an are covered with epoxy and serve as an absolute control. absolute control. 25mm Right: After functionalization, the Right: After functionalization, the chip is returned to Stanford chip is returned to Stanford University for measurements. During University for measurements. During the prototype experiments, the prototype experiments, macrofluidics macrofluidics are employed to are employed to repeatedly transport the reagents to repeatedly transport the reagents to and from the chip. Not shown are the and from the chip. Not shown are the readout electronics and magnetic readout electronics and magnetic field generators for the chip. field generators for the chip. While the prototype chips reach maturity, we also work to While the prototype chips reach maturity, we also work to to to ensure that the functionalization protocol and experimental ensure that the functionalization protocol and experimental procedure can be quickly migrated to a mass procedure can be quickly migrated to a mass - - produced version of the MagArray. This effort encompasses three produced version of the MagArray. This effort encompasses three critical critical areas: Whole areas: Whole - - wafer robotic biofunctionalization, alignment wafer robotic biofunctionalization, alignment - - and topography tolerant and topography tolerant microfluidics microfluidics for reagent transport, for reagent transport, and mass and mass - - producible producible microfluidic microfluidic interconnects that do not require the drilling of holes. All th interconnects that do not require the drilling of holes. All th ese three areas are designed ese three areas are designed to become integral parts of the manufacturing process. to become integral parts of the manufacturing process. Whole Whole - - Wafer Robotic Biofunctionalization Wafer Robotic Biofunctionalization Mass Mass - - Producible Producible Microfluidic Microfluidic Interconnects Interconnects Sensors 1-16: DNA or Antigen1 Sensors 49-64: Bare or Antigen2 The Prototype MagArray4 Chip The Prototype MagArray4 Chip Representative Functionalization and Representative Functionalization and Experimental Protocol Experimental Protocol Magnetic nanoparticle w/ Streptavidin Antigen 1: Ferritin Biotin Antibody (Anti-Ferritin) Antigen 2: BSA Nanoparticle Solution Sensors 17-48 Sensors 49-64 Sensors 1-16 Epoxy Antibody Solution Sensors 17-48 Sensors 49-64 Sensors 1-16 Epoxy BSA Solution Ferritin Solution Sensors 17-48 Sensors 49-64 Sensors 1-16 Epoxy Functionalization 1. The antigens are applied selectively Measurement Results Measurement Results Shown on the right is a measurement curve that was Shown on the right is a measurement curve that was recorded as MACS recorded as MACS nanoparticles nanoparticles , coated with , coated with streptavidin streptavidin , , were binding to a MagArray4 chip. Some sensors on the were binding to a MagArray4 chip. Some sensors on the chip were bare. These resulted in the yellow signal curve chip were bare. These resulted in the yellow signal curve with a final value of ca. 1 with a final value of ca. 1 μ μ V. V. Other sensors on the same chip were functionalized with Other sensors on the same chip were functionalized with single stranded single stranded biotinylated biotinylated DNA. These sensors, in red, DNA. These sensors, in red, show a rapid increase in signal to ca. 38 show a rapid increase in signal to ca. 38 μ μ V as soon as the V as soon as the nanoparticles nanoparticles are applied around t = 25 minutes. Thus, the are applied around t = 25 minutes. Thus, the nanoparticles nanoparticles are clearly revealing the presence of DNA on are clearly revealing the presence of DNA on the chip with a signal to noise ratio of ca. 31 dB. The signal the chip with a signal to noise ratio of ca. 31 dB. The signal rise time of the red curve can also give insights into the rise time of the red curve can also give insights into the biotin biotin - - streptavidin streptavidin binding kinetics. binding kinetics. Note that the washing the chip with Note that the washing the chip with deionized deionized water (H2O) water (H2O) leads to temporary shifts in the signal, but it does not leads to temporary shifts in the signal, but it does not remove the remove the nanoparticles nanoparticles . The wash at t = 73 minutes only . The wash at t = 73 minutes only minimally reduces the signal minimally reduces the signal - - MACS are bound strongly. MACS are bound strongly. In the second figure, a different functionalization based on In the second figure, a different functionalization based on antibodies was used. The red curve shows the binding of antibodies was used. The red curve shows the binding of nanoparticles nanoparticles to the to the ferritin ferritin - - functionalized sites. The large functionalized sites. The large signal increase of these sites reveals the presence of an anti signal increase of these sites reveals the presence of an anti - - ferritin ferritin antibody. On the other hand, the lack of a distinct antibody. On the other hand, the lack of a distinct signal increase of the BSA signal increase of the BSA - - functionalized sensors reveals functionalized sensors reveals that no anti that no anti - - BSA antibodies were present. BSA antibodies were present. The rise time of the anti The rise time of the anti - - ferritin ferritin signal is slower than in the signal is slower than in the experiment using DNA, possibly indicating a lower density experiment using DNA, possibly indicating a lower density of available biotin sites on the chip surface. The exact of available biotin sites on the chip surface. The exact relation of signal and binding kinetics is being investigated relation of signal and binding kinetics is being investigated in more detail. in more detail. The electron microscope images on the right, taken after the The electron microscope images on the right, taken after the experiment, show clearly that signal differences correspond experiment, show clearly that signal differences correspond to to nanoparticle nanoparticle coverage differences. coverage differences. Substrate SV SiO 2 SiO 2 Au Au lead Au lead Spotting Needle Upper Right: Upper Right: Shown here is the result of a sample Shown here is the result of a sample run of our functionalization robot. run of our functionalization robot. The robot has placed different The robot has placed different functionalizations functionalizations on different on different sensors as programmed with good sensors as programmed with good accuracy. accuracy. Lower Right: Lower Right: Shown here is a Shown here is a microfluidic microfluidic MagArray4 chip. Instead of the open MagArray4 chip. Instead of the open well that the prototype has, this chip well that the prototype has, this chip features closed features closed microfluidic microfluidic structures that carry the reagents structures that carry the reagents across the chip. Some of the larger across the chip. Some of the larger microfluidic microfluidic features are visible in features are visible in the center of the chip. Note also the the center of the chip. Note also the three three “ “ DNA Align DNA Align ” ” marks that aid marks that aid the robot the robot - - to to - - chip alignment. chip alignment. The robotic functionalization is The robotic functionalization is applied first, and then encapsulated applied first, and then encapsulated by the by the microfluidic microfluidic structures. structures. 400μm 12mm Left: Left: A schematic representation of the A schematic representation of the chip and the robotically controlled chip and the robotically controlled spotting needle used for the spotting needle used for the functionalization. functionalization. In a custom In a custom - - build application, the build application, the functionalization for each of the 64 functionalization for each of the 64 (or more) sensors can be individually (or more) sensors can be individually programmed. A video camera is used programmed. A video camera is used to control the functionalization and to to control the functionalization and to aid in the alignment of the robotic aid in the alignment of the robotic applicator to either a single chip, or applicator to either a single chip, or to an entire wafer. The program is to an entire wafer. The program is highly flexible and adaptable to a highly flexible and adaptable to a variety of chips. It can apply up to variety of chips. It can apply up to 384 different 384 different functionalizations functionalizations – – e.g. e.g. antibodies or genes of interest antibodies or genes of interest – – to an to an arbitrarily sized and positioned arbitrarily sized and positioned substrate. substrate. Shown in the lower image is the Shown in the lower image is the robotic applicator and video camera robotic applicator and video camera during a test run. during a test run. Topography Topography - - and Alignment and Alignment - - Tolerant Tolerant Microfluidics Microfluidics Optical microscope and electron microscope images of the two microfluidic layers combined and separated. The reagents are guided towards the sensor in the large and, since they are on a separate wafer, coarsely aligned PDMS channels. The reagents then traverse the actual sensor in the much narrower and precisely aligned SiO2 channel. Layer B: SiO2 Microfluidics Flow 20 μm To be suitable for high To be suitable for high - - yield mass production, it is important that yield mass production, it is important that the the microfluidics microfluidics are somewhat tolerant to small alignment and are somewhat tolerant to small alignment and process variations. This is achieved with a 2 process variations. This is achieved with a 2 - - layer process. Layer A layer process. Layer A carries large channels and is fabricated on a separate wafer fro carries large channels and is fabricated on a separate wafer fro m m polydimethylsiloxane polydimethylsiloxane (PDMS). Layer B is fabricated right on the (PDMS). Layer B is fabricated right on the MagArray4 substrate wafer from SiO2 with photolithographic MagArray4 substrate wafer from SiO2 with photolithographic precision. Layer B contains the small precision. Layer B contains the small microfluidic microfluidic features, such as features, such as sub sub - - micron channels over the active sensing elements. The overlap micron channels over the active sensing elements. The overlap of the layers is generously sized to accommodate alignment error of the layers is generously sized to accommodate alignment error s s (see finished sample structure on the right). (see finished sample structure on the right). After the MagArray4 substrate wafer has been robotically After the MagArray4 substrate wafer has been robotically functionalized, the support wafer carrying layer A is cold seale functionalized, the support wafer carrying layer A is cold seale d to d to the substrate wafer. Since PDMS is the substrate wafer. Since PDMS is elastomeric elastomeric , it can seal over , it can seal over uneven topography and even accommodate an occasional particle uneven topography and even accommodate an occasional particle – – this would be impossible with the standard procedure of anodic this would be impossible with the standard procedure of anodic bonding. bonding. Sensor Electric lead Connecting the Connecting the microfluidic microfluidic structures to the outside world is one of the engineering structures to the outside world is one of the engineering challenges. We have developed an automatable approach which does challenges. We have developed an automatable approach which does not require the drilling not require the drilling of holes into any of the wafers, which would be laborious and un of holes into any of the wafers, which would be laborious and un wanted for wafer processing. wanted for wafer processing. The main feature consists of cleavable edges that, once removed, The main feature consists of cleavable edges that, once removed, provide openings to the provide openings to the microfluidics microfluidics . These openings can be robotically sealed to a complementary, s . These openings can be robotically sealed to a complementary, s elf elf - - centering centering microfluidic microfluidic holder, which can be a cheap mass holder, which can be a cheap mass - - producible plastic part. On the right, a producible plastic part. On the right, a MagArray prototype with working two MagArray prototype with working two - - layer layer microfluidics microfluidics and and microfluidic microfluidic holders is shown. holders is shown. Microfluidic Holder Microfluidic Channel IV Tubing Large channel, Ca. 10μm deep Ca. 2-100μm wide Sensor Shallow trench, Ca. 200nm deep Ca. 1-5μm wide Layer B SiO2, 200nm thick PDMS Sub-micron channel, Ca. 180nm deep Layer A Layer B Layer A PDMS 10μm thick Support Wafer (glass) Large channel, Ca. 10μm deep Ca. 2-100μm wide Substrate Wafer (silicon) Sensor Shallow trench, Ca. 200nm deep Ca. 1-5μm wide Layer B SiO2, 200nm thick PDMS PDMS Support Wafer (glass) Substrate Wafer (silicon) Sub-micron channel, Ca. 180nm deep Layer A Layer B Layer B Layer B Layer B Microfluidic Layer B PDMS Support Wafer (glass) Substrate Wafer (silicon) Layer A Layer B Partial Cut Layer B PDMS Support Wafer (glass) Substrate Wafer (silicon) Layer A Layer B Insert blade and twist edge off Layer B Microfluidic PDMS Support Wafer (glass) Substrate Wafer (silicon) Layer A Layer B Microfluidic opening Layer B Layer B PDMS Support Wafer (glass) Substrate Wafer (silicon) Layer A Layer B Adhesive Microfluidic Holder Inlet / Outlet DNA Detection (ssDNA w/ Biotin) on the MagArray4 Wafer RB2, Chip 2-2, Nov-29-2005, using Streptavidin Nanoparticles (MACS) -10 0 10 20 30 40 50 0 10 20 30 40 50 60 70 Timeline, Minutes Signal Amplitude, μV ssDNA-Biotin Bare Sensor H2O H2O H2O H2O Nanoparticles <-- SNR ~ 31 dB --> Figure 1: DNA Detection Antibody Detection (Anti-Ferritin w/ Biotin) on the MagArray4 Wafer RB2, Chip X-1, Jan-18-2006, using Streptavidin Nanoparticles (MACS) -10 0 10 20 30 40 50 0 10 20 30 40 50 60 70 Timeline, Minutes Signal Amplitude, μV Ferritin BSA PBS H2O PBS PBS PBS PBS PBS PBS PBS PBS H2O H2O Nanoparticles <-- SNR ~ 21dB --> Figure 2: Antibody Detection The measurement is carried out while the nanoparticles bind Functionalization 2. The biotinylated antibody is applied globally