Microfluidics Foundry

  

Quick Menu

 

Click to jump to sections: 

• Overview & Getting Started
• Technology

• Microfluidic Valve Technology
• Microfluidic Large Scale Integration
• Designing Your Own Device
• Available Valve Types
• Multi-Height Molds
• Chip Bonding
• Basic Design Rules
• Mask Design Rules
• Getting Started with AutoCAD
• Testing Your Device
• Testing Using Syringes
• Testing Using Manifolds
• Testing Using Pre-Made Controller Box
• Building Your Own Valve Controller
• Contacts and Location

 

  

Overview of Services

 

Welcome to the Stanford Microfluidics Foundry

 

We provide the following services:

• Fabrication of Custom Designed Microfluidic Chips Based on Microfluidic Valve Technology
• Fabrication of Custom Designed Replica Molds Used for Soft Lithography
• Fabrication of Pre-designed Microfluidic Chips
• Consulting on Microfluidic Design Using the Design Rules Outlined by the Stanford Microfluidics Foundry
• Instructions on Building Your Own Valve Controller for Testing Microfluidic Chips

 

 

Getting Started

 

• Stanford Users:  LOGIN or REGISTER for an iLab account using valid Stanford credentials (SUNet ID).

• Non-Stanford Users:  Click to LOGIN with approved iLab credentials. Or, click to SIGN UP for an iLab account.

 

• Visit the Customer Help Manual for further guidance using iLab's Core Facility Services.

 

 

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Technology

 

All microfluidic chips and molds produced by Stanford Microfluidics Foundry are based on multilayer soft lithography which enables integrated membrane valves and microfluidic large scale integration technology.

To learn more about this technology check out Microfluidic Valve Technology and Microfluidic Large Scale Integration. Also, check the video on multilayer soft lithography

 

Marcus, 2006	Hansen et al., PNAS, 2004.

 

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Microfluidic Valve Technology

 

Soft Lithography is a microfabrication process in which a soft polymer (such as polydimethylsiloxane (PDMS) ) is cast onto a mold that contains a microfabricated relief or engraved pattern. Using this technique, membrane microvalves can be produced. This membrane microvalve is the fundamental component which enables liquids to be controlled on-chip and is the key to realizing microfluidic large scale integration. 

A basic microfluidic device is composed of two elastomer layers. One layer contains channels for flowing liquids (flow layer), and the other layer contains channels that deflect the membrane valve into the flow channel and stop liquid flow when pressurized with air or liquid (control layer).

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Master Molds

Molds containing the relief of the desired microfluidic circuit are made using conventional photolithography. This entails first designing your desired microfluidic network in a CAD program and printing it onto a transparency film using a very high resolution printer. Next, an appropriate photosensitive polymer (photoresist) is spun onto a silicon wafer and ultraviolet light is exposed to the wafer through the overlaying mask. Finally, the wafers are developed to reveal the transferred microfluidic network pattern on the silicon wafer. Note: one mold is made for the control layer and one mold is made for the flow layer.

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Photoresists and Geometry of channels:

A photoresist is a light-sensitive material used to form a patterned coating on a surface.

Photoresists are classified into two groups: positive resists and negative resists.

• A positive resist is a type of photoresist in which the portion of the photoresist that is exposed to light becomes soluble to the photoresist developer. The portion of the photoresist that isunexposed remains insoluble to the photoresist developer.

• A negative resist is a type of photoresist in which the portion of the photoresist that is exposed to light becomes insoluble to the photoresist developer. The unexposed portion of the photoresist is dissolved by the photoresist developer.

We use SU 8 series negative photoresist to create a rectangular type channels since after hard baking features have rectangular profile.
To make rounded channels we commonly use AZ  and SPR positive photoresists and they have  rounded profile after hard baking.

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Single layer molds vs. multi layer molds: 

Most commonly used are molds that have one layer of photoresist and all features are the same height. 
Multi layer molds are made in cases when it is necessary to have features with different heights.

Schematic of a multi-height (layer) mold showing 3 layers of different heights.

 

In this case, a second layer of photoresist is applied to the first one, and all the same basic mold making steps are repeated except exposure. Before exposing, it is necessary to align the first layer with the mask of the second layer.

In order to precisely position the mask of the second layer with the first layer mold, both masks for layers one and two should have alignment marks on them.
For a three layer mold the same steps are applied, and all 3 masks for those layers must have alignment marks in order to work.

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PDMS Devices (Chips)

 

Types of devices
Push up	Push down
Control lines pass under the flow channels. Pneumatic pressurization of the control line causes a membrane to deflect up into the flow structure, sealing the channel. Deep reaction chambers may be integrated into the flow layer (upwards).	Control lines pass over the flow channels. Pneumatic/hydraulic pressure in the control lines flattens the membrane valve downwards to create a seal.

 

 

Steps to make devices:	Push up device	Push down device
Making Control layer	Spinning PDMS on control mold to form a thin layer and bake	Pour PDMS onto wafer to form a thick layer and bake
Making Flow layer	Pour PDMS onto wafer to form a thick layer and bake	Spinning PDMS on control mold to form a thin layer and bake
Aligning layers	Align flow on control layer	Align control on flow layer
Bonding layers	Bake both layers	Bake both layers
Bonding device to a substrate	Bond the device to a substrate to seal the control layer	Bond the device to a substrate to seal the flow layer

 

 

The following figure shows the basic fabrication process for this two-layer device (courtesy Dr. Carl Hansen): 

When a control channel and a flow channel cross, if the area of the intersection is large enough, a valve is created. The thin membrane separating the two channels deflects into the flow channel when the control channel is pressurized, creating a complete seal. The following picture shows a typical valve in the closed state (courtesy Dr. Carl Hansen): 

Marc A. Unger, Hou-Pu Chou, Todd Thorsen, Axel Scherer, and Stephen Quake, "Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography," Science, vol. 288, no. 7, pp. 113-116, April 2000. 
David C. Duffy, J. Cooper McDonald, Olivier J.A. Schueller, and George Whitesides, "Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane)," Analytical Chemistry, vol. 70, no. 23, pp. 4974-4984, December 1998.

 

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Microfluidic Large Scale Integration

 

Microfluidic large scale integration (mLSI) refers to the development of microfluidic devices with thousands of integrated micromechanical valves and control components. mLSI allows nanoliters or picoliters of liquid to be controlled and manipulated on-chip for a large number of applications including molecular biology, cell biology, etc.

 

 

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One of the most simple integrated microfluidic devices which can be created using membrane microvalves is a peristaltic pump. The pump is formed by designing three valves in series and activating the valves sequentially, see figure (courtesy Dr. Carl Hansen).

 

 

Other examples of components which enable large scale integration include microfluidic multiplexers, memory chips, etc. More information about mLSI, useful design components and design rule strategies to achieve large scale integration can be found in:

Melin J, Quake SR. "Microfluidic Large-Scale Integration: The Evolution of Design Rules for Biological Automation" Annu. Rev. Biophys. Biomol. Struct. 36:213-31, 2007.

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Check out the list of publications from the Quake Group for interesting mLSI examples. Very illustrative animations and movies demonstrating multilayer soft lithography are available at the web site of Fluidigm Corp. (make sure to check out the animated tutorial).

 

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Designing Your Own Device

 

To design your own microfluidic device, please follow the Basic Design Rules and Mask Design Rules and make use of all the design information and reference articles provided on our website. 

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If you have further questions, please contact Wei Gu at sufoundry@lists.stanford.edu.

 

 

Available Valve Types

 

When designing your microfluidic circuit, you should consider which type (or types) of valve is most appropriate for your design. 

There are four available valve types:

• Push-down

• Push-up

• Sieve valve

• Push-up and Push-down

   

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Push-down Valves

Configuration :
Control lines pass over the flow channels. Pneumatic/hydraulic pressure in the control lines flattens the membrane valve downwards to create a seal.  This geometry is suitable only for low aspect ratio (1:10), shallow (approx 10um) flow-structures and does not allow for deep reaction chambers to be integrated on the flow layer.

 

 

Standard Flow Geometry :  
Standard flow features for the push-down valve is 100um wide by 13um tall.  

Standard Control Geometry :  
Standard control channels are 100um wide and either 10um or 25um tall. This results in a valve junction area of 100um * 100um.

  
Applications : 
This geometry is particularly well suited to applications where the flow structure must be in direct contact with the substrate (e.g. spotting DNA, patterned substrate, etc.)

Sealing to Substrate :  
The chip flow structures may be sealed either directly onto a glass cover-slip / glass slide, or onto a glass substrate coated by a thin layer of PDMS.  Devices sealed to glass are only rated to approximately 6psi flow pressures before delamination occurs.  Devices sealed with a third layer of PDMS are rated to 20psi flow pressure. The chip may also be bonded to a glass substrate directly via plasma bonding.

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Push-up Valves

Configuration : 
Control lines pass under the flow channels. Pneumatic pressurization of the control line causes a membrane to deflect up into the flow structure, sealing the channel. Deep reaction chambers may be integrated into the flow layer (upwards).  

 

 

Standard Flow Geometry : 

• Shallow Channel Option : Flow features are similar to push-down valves (100um wide, 13um tall). 
• Deep Channel Option : Maximum flow channel heights at a valve junction is approximately 45um. For 45um tall flow channels, a valve junction area should be 200um * 200um.

Standard Control Geometry :  

• Shallow Channel Option :  Standard control channels are 100um wide and either 10um or 25um tall. This results in a valve junction area of 100um * 100um.    
• Deep Channel Option :  Control channels are 200um wide and 25um tall.  For 45um tall flow channels, a valve junction area should be 200um * 200um.

Applications : 
Deep channel push-up valves are suitable for applications that require suspensions of large particles (eukaryotic cells, large beads ...). 

Sealing to Substrate :  
It is recommended to bond this type of chip to a glass substrate coated with a thin layer of PDMS or directly to a glass substrate via plasma bonding.

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Seive Valves

Configuration : 
Sieve valves are different from push-down and push-up valves in that they intentionally do not create a tight seal. The flow channel has a rectangular profile instead of a rounded profile. When the valve is closed, the edges of the valve are not sealed and allows liquid to pass through along the channel edges. Most commonly, sieve valves are of the ‘push-up variety' (the control channels pass underneath the flow channels) and the membrane deflects upwards.

 

 

 

 

Applications:
Sieve valves are useful for creating affinity columns of beads by blocking the movement of beads, but allowing reagents to flow through. The beads can then be released by opening the valve and allowing them to flow to another part of the chip. The sieve valve may also be used for filtering purposes. 

Sealing to Substrate :  
It is recommended to bond this type of chip to a glass substrate coated with a thin layer of PDMS or directly to a glass substrate via plasma bonding.

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Push-up and Push-down Valves

Push-up and Push-down valves are a combination of both types of valves and are made when a control channel lies between two flow channels.

 

 

  

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Multi-Height Molds

 

The standard microfluidic device using membrane valves includes designing a control mold which has control channels of height A and a flow mold which has flow channels of height B. However, some applications require channels and features to have various heights on the same mold. For example, a reaction chamber may need to be 100um tall, however the tallest valvable flow channel is only 45um.

 

Schematic of a multi-height (layer) mold showing 3 layers of different heights.

The Stanford Microfluidics Foundry allows molds with up to 3 layers of complexity to be fabricated. Below are design rules which should be followed if your design requires more than a single layer height.

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The most common sequence of multi-height molds are:

Layer 1: Rounded Cross-sectional Profile 
Layer 2: Rectangular Cross-sectional Profile 
Layer 3: Rectangular Cross-sectional Profile

 

In a multi-height mold, the first layer has to be rounded and the rest must be rectangular layers. In cases when all design features on a multi height mold need to be rectangular (made with negative SU8), a "Blank" Layer is used.

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"Blank" Layer

A "Blank" layer is a layer on the mold that is made with positive photoresist and does not have any features or channels on it, but does have alignment marks. Thus, multiple negative layers of SU 8 photoresists can be aligned using positive layer marks. 
In general, the first layer of a multi height (layer) mold is made with positive resist, and all consecutive layers with negative, because of the physical properties of both resists.
In order to align mask of the second layer with the first layer wafer, it is important that the first layer can be seen through the second spin coated layer.
Negative resist, such as SU8, is a clear viscous liquid. Positive resist, such as AZ or SPR has an amber-red color. 
Only when the first layer is positive, can it be seen though spin coated layers of SU 8, allowing the layers to be aligned.

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Alignment Marks

Alignment marks are very important in multi-height (layer) molds.

If you are designing a 2 layer mold:
Use the AutoCAD file of alignment marks (hybrid_mold_align.dwg)
Layer names: Rounded Layer1, Rectangular Layer2

If you are designing a 3 layer mold:
Use the AutoCAD file of alignment marks (hybrid_mold_align.dwg)
Layer names: Rounded Layer1, Rectangular Layer2, and Rectangular Layer3

When designing a mold having shallow channels connecting significantly taller features, ensure that the shallow channels are designed such that they can be fabricated before the taller features, i.e. shallow channels = Layer 1, taller features = Layer 2 (see Picture above). 

When designing a mold having features directly on top of another layer of features, ensure that enough alignment tolerance is given (i.e. 30um in all directions).

 

When designing multi-level molds with significant differences in heights, special considerations must be taken. If your design resembles Figure 2 where Layer 2 is much taller than Layer 1, the PDMS membrane thickness may become very uneven resulting in non-functioning valves.

Schematic of short channels connecting tall features where valves are desired at the short channels. Push down type device.

 

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Chip Bonding

 

When microfluidic devices are ready, in some applications, it is required that they are bonded to the substrate (usually glass). As an example, in Figure 1, the flow layer gets sealed after bonding the device to the glass:

 

 

Schematic of a Push down device bonded to the glass slide. PDMS to PDMS type of bonding. 
Stanford Microfluidics Foundry is able to bond PDMS chips to glass substrates (i.e. glass slides, cover-slips or chips) or silicon substrates (i.e. Si chips).

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There are three main methods for bonding your PDMS chip to a substrate:

Direct Thermal Bonding to Substrate

• This method requires your substrate to withstand 80C. Devices sealed to glass are only rated to approximately 6psi flow pressures before delamination results. 

 

Bonding To Substrate Coated with PDMS

• For this method, your substrate is coated by a thin layer of PDMS and the PDMS chip is directly bonded to this surface. Devices are rated to 20psi pressure.

 

Direct Bonding Via Oxygen Plasma

• The chip may also be bonded to a glass substrate directly via plasma bonding and is rated to at least 20psi. If any part of your chip which may be at risk for collapses to occur, plasma bonding is not recommended.

 

Here is a list of Available Coverslips and Glass Slides provided by the foundry. 
If you don't see the type and size you need, you would have to provide your own.

 

Coverslip Size (mm)	Thickness
	# 1	# 1.5	# 2
18x18	Y	Y	Y
22x22	Y	Y	Y
22x30	Y	Y	-
22x40	Y	Y	-
22x50	-	Y	-
22x60	Y	Y	-
24x30	Y	Y	-
24x40	Y	-	-
24x50	Y	-	-
24x60	-	Y	-
25x25	Y	Y	Y
25x50	-	-	Y
35x60	Y	-	-
43x50	Y	-	-
48x65	Y	-	-

  

Available Glass Slides

25x75 mm
50x75 mm

 

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Basic Design Rules

 

Here are the basic design rules for microfluidic devices fabricated at the Stanford Microfluidic Foundry. Following these rules for a design improves the chances that the devices will be easily manufactured and operational. 
It is assumed that the reader has an understanding of the basic Microfluidic Valve Technology fabrication process.

For information on design strategies in microfluidic large scale integration, and various microfluidic control subcomponents, please refer to the following publication:

 

J. Melin, S.R. Quake, Microfluidic Large-Scale Integration: The Evolution of Design Rules for Biological Automation, Annual Reviews in Biophysics and Biomolecular Structure 36:213-31, 2007

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Basic Design Rules 

1. Please refer to the AutoCAD template file when starting your design.

It is recommended that you use this file since units and layers are already defined. Refer to Mask Design Rules for more detailed guidelines for your layout.

2. All devices must have standard Device Alignment Marks.

Device alignment marks are different from mask alignment marks and they are for aligning different PDMS layers to each other.  These alignment marks can be downloaded here for a standard two layer device. This set of alignment marks must be copied and placed on the left and right edge of the wafer layout. Subsets of crosses (alignment marks) should also be included near the most critical chip features, and marks at the periphery of the device (four corners).  If possible, a string of alignment marks along a chip edge is useful.

If your design requires multiple height molds, please read the guidelines and alignment mark requirements for Multiple Height Molds.

3. Standard input/output holes to access the flow and control channels punchers.

All punch locations must be clearly marked with a standard punch marker. The foundry can punch holes of the following sizes (note that 20 gauge is standard).

If other punch hole sizes are required, you must provide this hole puncher yourself. 

Gauge #	Approximate Diameter
14	1.75mm
15	1.63mm
20	0.66mm
25	0.36mm

 

You must include standard hole markers where you want holes to be punched. These markers can be copied from the AutoCAD file std-punch-align-marks.dwg. 

 

4. No structure (control or flow) can be fabricated having an aspect ratio lower than 1:10 (height : width).  

Structures with lower aspect ratios are prone to collapse.  If a feature is wider than this design rule permits, support posts must be added to ensure the aspect ratio is no lower then 1:10 in between posts (i.e. posts every 100um for a 10um high structure). If your device does not call for valves (i.e. it is a simple single layer device), it is not as prone to collapses and lower aspect ratio can be used.

5. All devices must be designed with a border indicating where the chip should be diced. 

6. Designs must incorporate sufficient tolerances to allow for easy layer/layer registration.  Designs must account for alignment tolerances of 30um.  This means that the device must be designed to function properly despite 30um errors in alignment in all directions. 

7. Crossovers of control lines and flow lines can be created without resulting in a valve at each crossover point. To achieve this, the control line should be designed narrower (15-30um) at these crossover points to ensure no membrane valve is formed.

8. The following table indicates a nominal valve area for various channel heights (both push-down and push-up designs) and an approximate closing pressure:

Push-down Valve			Push-up Valve
Valve Area	Flow Channel Height	Closing Pressure	Valve Area	Flow Channel Height	Closing Differential Pressure
100um x 100um	5um	10psi	100um x 100um	5um	5psi
100um x 100um	10um	10psi	100um x 100um	10um	5psi
100um x 100um	15um	10psi	100um x 100um	15um	5psi
N/A	20um	N/A	100um x 100um	20um	5psi
N/A	25um	N/A	150um x 150um	25um	5psi
N/A	30um	N/A	150um x 150um	30um	5psi
N/A	35um	N/A	150um x 150um	35um	5psi
N/A	40um	N/A	200um x 200um	40um	5psi
N/A	45um	N/A	200um x 200um	45um	5psi

 

Note: Push-down valves which seal reliably within a reasonable pressure range do not have flow channel heights exceeding 15um.

9. The following table summarizes some critical design rule information that is useful when designing your device:

Parameter	Value
Minimum overall chip thickness	3mm
Maximum overall chip thickness	7mm
Minimum flow channel height ( push-down valve )	5um
Maximum flow channel height ( push-down valve )	15um
Minimum flow channel height ( push-up valve )	5um
Maximum flow channel height ( push-up valve )	45um
Minimum possible feature width	15um
Maximum possible feature height (rectangular profile only)	150um
Nominal control channel height	10um or 25um
Minimum spacing between borders of adjacent devices	2mm
Minimum center-to-center spacing between punch holes (for 20 gauge)	1500um
Minimum center-to-center spacing between punch holes (for 15 gauge)	2000um

 

 

 

 

10. When designing control channels layout, control channels which are 10um tall should be spaced at least 15um apart. Control channels that are 25um tall should be spaced at least 40um apart.

11. Largest aspect ratios possible for rectangular structures is approximately 1:1 (Y:X, Y=height of feature, X=width of feature). For features which are very close together the largest aspect ratio is 1.5:1 (Y:X, Y=height of feature, X=spacing between features).

12. No more than 35 hole punches will be allowed per chip except for certain pre designed chips.

 

 

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Mask Design Rules

  

Masks for generating the molds for soft lithography are commonly designed using AutoCAD and then printed onto transparencies films. The following are design rules you should follow when preparing your microfluidic circuit before submitting it to Stanford Microfluidics Foundry. Also, be sure your design follows the Basic Design Rules.

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Mask Specifications 

1. Start by using the  mask template provided. This template corresponds to a 4" diameter silicon mold size (recommended size).

2. AutoCAD files should be submitted to the foundry in .dwg format using the version AutoCAD 2000, 2004, or 2007.

3. Ensure that AutoCAD is set to the correct internal units before beginning your design. Under the menu Format -> Units, ensure the following settings: 

Length Type: Decimal, Length Precision: 0.0000

Drag-and-drop Scale: Microns

4. Masks can be printed at two approximate resolutions, which results in two different minimum feature sizes possible: 
20 000dpi => recommended minimum feature size 15um
40 000dpi => recommended minimum feature size 10um

5. Each mold (i.e. control and flow) is drawn in a separate AutoCAD layer. If you are creating hybrid molds where you have multiple photoresist heights on a single mold, each photoresist height must be drawn in a separate layer.

6. The AutoCAD file which you submit to the foundry must have all layers in one single file. The layers should be drawn and overlapping as corresponding to your design (you should not separate out individual layers in different areas of the workspace).

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General Layout

1. Try to fit in as many devices as possible onto a single mold. However, ensure there is enough space between chips for adequate bonding and to enable the PDMS to be cut into individual chips (see  Basic Design Rules). 

2. Leave a ring of approximately 0.5cm free around the mold perimeter. This area should be void of any design or critical chip area since photoresist uniformity is least reliable around the perimeter of a wafer.

3. Each layer should have lines or right angle corners indicating where the PDMS should be cut around the perimeter for the devices. Make these cut lines approximately 50um wide. 

4. Masks are printed on 8.5" x 11" transparency sheets. Therefore, a total of four molds in four quadrants can fit on each sheet. If you have several chip versions which do not fit onto one mold, design a second mold in another quadrant.

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Labeling

1. All layers should have text labels clearly identifying:

Your Name 
Date 
Mold Name 
Layer Name (for mulit-height molds, there will be multiple layer names for each mold) 
Project Name 
Version Number

 

All these labels should be within the perimeter of each mold. The height of the characters in the labels should preferably be such that the letter "L" is at least 650um high, if space is available.

2. Polyline-based text can be created by copying and pasting (and scaling appropriately) individual characters from either one of these files: alphabet.dwg (AutoCad 2004 format), or alphabet.dxf (AutoCAD R12 format). You can also use the linetext AutoLisp application to draw text (instructions on how to use the application are in the linetext-readme.txt file included with the application).

3. If you have several chips of different designs on one mold, label each chip by a different name or number, i.e. Chip1, Chip2, etc.

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Feature Specifications

• All features should be composed of closed polylines.
• No hatched or filled in features should be created.
• All polylines or regions that form a single pattern must touch each other to avoid any gaps between them in the printed mask (i.e. an unintentional gap of 2um could cause a catastrophic blockage in your fluidic channel). The polylines and regions should not cross each other (use "union" function of AutoCAD if necessary).
• Any pattern corresponding to a thick layer of PDMS that is peeled off the mold for subsequent alignment and bonding onto a patterned thin layer will be scaled up by 1.5% to compensate for the shrinkage of the PDMS after peeling. However, this will be done by the foundry – do not do this scaling yourself.

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Alignment Marks

Mask Alignment Marks for Multi-Height Molds

• Sets of alignment marks for aligning between multiple layers of photoresist on a single mold should be spaced apart by approximately 6.5cm on a 4inch wafer. 
• Alignment marks for layers which are composed of positive photoresist should be created using positive_alignment_marks.dwg. Alignment marks for layers which are composed of negative photoresist should be created using negative_alignment_marks.dwg. The positive alignment marks should be centered in the negative alignment marks.

Device Alignment Marks for PDMS-PDMS Alignment 
Several alignment marks should be placed around the device area.

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Getting Started with AutoCAD

 

The following gives some useful guidelines for using AutoCAD to create your own microfluidic designs. AutoCAD is a comprehensive software package, but for purposes of getting started quickly only a select number of instructions are given here, which can be used in creating 2D designs for microfluidic circuits. These guidelines are meant for those who have never used or are only vaguely familiar with AutoCAD.

Have fun designing your microfluidic circuit!!

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1.0 Getting Started

Working in AutoCAD can be done using command line instructions, point-and-click commands, menu selections, or a combination of all three. Functions are often duplicated between each style of working - many command line instructions can be found in the menus, etc.

1.1 Layers

To create objects/shapes in AutoCAD start by selecting an appropriate layer to draw it in. The pull-down layer menu is most likely found in the top right of the AutoCAD window and lists the layer name, color, if the layer is active/displayed (light bulb is on), and if the layer is locked or not (if it is locked you are not able to modify this layer). To create a new layer or modify an existing one, go to Format->Layer. When designing the microfluidic circuit, a separate layer should be used for each resist layer in your design, i.e. control, flow_rounded, flow_nonrounded, etc. There are 3 other pull-down menus related to the main layer menu found to the left on the screen. The 'ByLayer' option should be selected in these 3 menus so that the correct attributes for each layer are represented in the drawing window.

1.2 Units and Precision

Before you start your design, ensure that the internal units and precision is set up properly. Go to Format -> Units and make sure the Precision is set to 0 and Scale/Units is set to 'microns'.

1.3 Creating Shapes

Once you have defined and selected the appropriate layer, you can start to draw your design. One way to do this is to choose the desired shapes from the panel menu on the left of the screen, i.e. circles, lines, rectangles, polygons, etc. After clicking on the desired shape and clicking in the drawing window to initiate the shape, you can either simply terminate the shape by clicking in the drawing window or use the command line to specify quantitative details for the shape. For example, a circle can be drawn by clicking on the circle in the panel, clicking in the drawing window to define its midpoint, and then typing the desired radius in the command line.

1.4 Miscellaneous

When working in AutoCAD, be sure the 'Model' tab is selected on the bottom part of the drawing window. When saving an AutoCAD file, save it as a .dwg or .dxf format.

 

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2.0 Drawing Tools

2.1 Measuring Objects

To measure the length or area of a shape you simply use the ruler tool located as a point-and-click option on the top left of the window. You may also use the menu option to access this tool Tools->Inquiry->Distance or Tools->Inquiry->Area. If you want to measure length, activate this tool and click the length you want to measure in the drawing window. If you want to measure area, activate the area tool and click on the vertices of the shape you are measuring, press enter to get the final area result.

2.2 Changing Layers of Shapes

If you want to change the layer of an existing shape, simply select the shape and go to the main layer pull-down menu and click the desired layer. The shape layer will change immediately. Keep in mind that if the chosen layer is not active (light bulb is off) these shapes will not be displayed in the drawing window until you activate this layer by turning on the light bulb (clicking on the bulb).

2.3 Helpful Options

Some helpful options are located at the bottom of the drawing window including the ORTHO and OSNAP options. The ORTHO option allows you to draw straight lines or move shapes in a straight line easily and snaps to the closes 90 degrees. The OSNAP option enables you to accurately select vertices on shapes and lines, making it easier to align and join objects at specific locations, etc. Once OSNAP is active and you select a shape to draw, dragging the curser across existing shapes reveals specific vertices (indicated by yellow squares) and midpoints (indicated by yellow triangles) clearly.

2.4 Creating Text

It is very useful to label your microfluidic circuit with information such as project number, version number, date, etc. Instead of drawing shapes to create individual letters, a Lisp script has been created which allows you to generate letters via the command line in AutoCAD. First, in an internet browser go to http://thebigone.stanford.edu/foundry/maskdesignrules.htm and click on the 'linetext' hyperlink to download the Lisp script as well as a linetext-readme.txt file of how to use the script. Save these files in an appropriate directory. To create text in your AutoCAD design go to Tools->AutoList->Load and browse your directories until you find the saved Lisp file. Then click 'Load' and close the window. In the AutoCAD command line enter '(linetext)'. The program will first ask you to enter a location for the text; click in the drawing window where you want to initiate text. Then enter the text you would like to write in the command line. Press enter and the text will appear in the desired location. The text will be quite small so you will need to zoom in to see it and also scale it up to an appropriate size.

Note: before using the linetext program, ensure that the ORTHO and OSNAP options at the bottom of the drawing window are deactivated, otherwise the text created will be distorted.

 

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3.0 Manipulating Objects

3.1 Moving

To move a shape in the drawing window, select the shape by clicking on it or drawing a select box around it using the left mouse button. Then right click and choose 'Move'. Click on the shape and move it to the desired location. You can also specify the number of microns you would like to move it by initiating the move in the desired direction (having the ORTHO option active at this time is recommended) and then typing the number of microns in the command line.

3.2 Mirroring

To mirror a shape, select the shape and then choose the point-and-click mirror button or use the menus Modify->Mirror. The command line will ask you to specify the first point of the mirror line. Click to define the mirror line (it is recommended to have the ORTHO option active at this time). Then the command line will ask if you want to delete the source object. If you simply want to mirror the existing shape type 'Y', but if you want to produce a copy of the existing shape type 'N'.

3.3 Rotating

To rotate a shape, select the shape and then choose the point-and-click rotate button or right click and choose Rotate. The command line will ask you to specify the base point or rotation point – click on the shape to indicate this point (it is recommended to have OSNAP active at this time to quickly be able to select specific vertices). Then type the desired angle of rotation in the command line.

3.4 Scaling

To scale the size of a shape, select the shape, then right click and select scale. The command line will ask you to specify the base point from which scaling should occur. Click on the desired base point (it is recommended to have OSNAP active at this time to quickly be able to select specific vertices). Then type the desired scaling factor, i.e. if you want to increase the size of the shape by 1% type 1.01, if you want to decrease the size of the shape by 10% type 0.90.

3.5 Arraying

Many times it is useful to copy a specific shape a number of times with predefined equal spacing between each copy. This can be done in one axis (X or Y) to create an array or in both axes to create a matrix. Select the desired shape and go to Modify->Array and choose the number of rows and column you would like to produce of this shape. Select if you want a rectangular or polar array (rectangular array most common). Indicate the desired row and column offset (remember to add the width and length of the shape if you do not want the shapes to overlap). Note that a positive row offset adds rows upwards and a positive column offset adds columns to the right (specifying negative offset numbers adds rows and columns in the opposite directions). Specify the angle of the array in degrees (for a conventional rectangular array this would be zero).

3.6 Unionizing

Shapes can be unionized to become one single shape. When drawing in AutoCAD, shapes and lines will be composed of 'polylines' or 'lines'. To verify this you can find the properties of a particular shape by selecting it, right clicking, and selecting properties. Once you are satisfied with your shape, you are able to join or subtract it with/from another shape. However, to do this you must change the shape property to 'region' by typing 'region' in the command line, clicking on your shape, and pressing enter. Verify this change by checking the shape property again. To join two or more shapes together, ensure they are all regions. Then type 'union' in the command line, click on the desired shapes, then press enter. The shapes should now be joined to form a single region. Before unionizing, make sure that the shapes are overlapping or clearly joined by their vertices to avoid unwanted, almost undetectable gaps between shapes.

3.7 Subtracting

Subtracting shapes from each other can be useful to create custom shapes. The procedure is very similar to unionizing shapes. First, the specific shapes must be regions (see Section Unionizing to learn how to do this). Then type 'subtract' in the command line, enter, select shape 1, enter, select shape 2, enter. This sequence results in shape 1 – shape 2 = shape 3.

3.8 Object Properties

To know the properties of a specific shape, click the shape, then right click and select properties. In the properties window you are able to change shape properties such as layer, line type, line weight, the coordinates of specific vertices, the length of a line, etc.

3.9 Zooming

To zoom on the drawing window, simply use your middle mouse scroll button to zoom in or out. You may also use the point-and-click zoom in and zoom out buttons on the window panel. 

 

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Testing Your Device

 

So now you have designed your microfluidic circuit, received your chip and are ready to start testing your microfluidic device!

Your test set-up will of course be specific to your application, but there are a few techniques outlined here which you may want to use to get started or which you may find useful to integrate into your particular testing set-up.

 

1) Testing Using Manually Controlled Syringes (parts list included)

2) Testing Using Manually Controlled Manifolds (parts list included)

3) Testing Using Pre-made Controller Box (purchasing info included)

4) Building Your Own Valve Controller (parts list and assembly instructions included)

 

For most designs, valve functionality can be tested by first filling the control lines with DI water. Due to the gas permeable properties of PDMS, the dead-end control channels will be depleted of air (air will diffuse through the PDMS) if you continue to push liquid into the control channels resulting in a bubble-free liquid filled channel. Once the channels are filled, if you apply further pressure you will be able to close the on-chip valves. The opening and closing can be monitored under a microscope. The figure below shows a typical open and closed valve. Anywhere between 0-30psi may be required to close the on-chip valves.

 

 

 

Fluidigm

 

Top photo shows a control line (horizontal) and a flow line (vertical) crossing at 90° and the valve is open. Bottom photo shows the valve closed when pressure is applied to the control line.

  

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Testing Using Syringes

 

You can simply use manually controlled syringes to pressurize control and flow lines by hand. Figure 1 shows a schematic of a set-up where plastic syringes are connected to the chip control and flow ports via Tygon tubing connected to hollow steel pins. Note that precisely controlling the pressure applied to the liquid via the manual syringes is very difficult (you may be applying a great amount of pressure onto the liquid while you may feel like you are not pushing the syringe very hard).

 

 

 

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Testing Using Manifolds

  

So now you have designed your microfluidic circuit, received your chip and are ready to start testing your device. You can build a set-up where pressure can be switched between control lines and between flow lines manually. If you have a continuous flow air supply, you can build a set-up where you regulate the pressure supply (in the range 0-30 psi) to several independent manifolds. Each manifold allows you to manually control the supply pressure (ON/OFF) to several independent control lines or flow lines.

 

Figure 1 shows a schematic of such a set-up where two pressures (P1, P2) are regulated from a single pressure source (P0). Each regulated pressure line is connected to a separate manifold comprised of 5 manually controlled outputs. One manifold is connected to the on-chip control lines and the other manifold to the on-chip flow lines.

 

 

 

Figure 1.  Schematic of fluidic control set-up using regulated pressure and manually controlled manifolds.

 

 

Parts List
Prerequisites	Inverted microscope / stereomicroscope
	Constant air supply
Materials Needed for Fluidic Connections	Disposable stainless steel dispensing needles to connect to syringe 23 gauge, 0.5" long, type 304, ID 0.017", OD 0.025” (Supplier: McMaster-Carr, Santa Fe Springs , CA , USA )
	Tubes for liquid/gas
	Tygon tubing (flexible plastic tubing for fluidic connections), ID 0.02", OD 0.06", 500ft, 0.02" wall (Supplier: VWR, Brisbane, CA, USA)
	Steel pins for chip-to-tube interface 0.025 OD x 0.017 ID, 0.500" length, s/s tube, type 304, cut, deburred, passivated (Supplier: New England Small Tube, Litchfield , NH , USA )
Materials Needed for Pressure Regulation and Control	Pressure regulators 0-30psi, Airtrol Inc., part number R-800-30-W/K (search for a local distributor at www.airtrolinc.com )
	Pressure gauges 0-30psi, Noshok brand, part number 15.100.30KPA (distributor for pressure regulators should have similar gauges available)
	Luer manifolds (2 to 5 ports), from Cole Parmer, part numbers C-06464-80, C-06464-82, C-06464-84, C-06464-86.
	Fittings to connect regulators to gauges and to luer manifolds: Supplier: Cole Parmer Part numbers: C-06349-20 (nylon tee), C-31200-60 (female luer to 1/8" NPT)
	Fittings of your choice to connect regulators to your compressed air supply.
	¼" OD x 1/8" ID Tygon tubing (Swagelok, part number RLT-2-4)
	Various other fittings and things that are useful:From Cole Parmer: Male luer lock 3-way stopcock (cat# C-30600-02), microbore Y connector (cat# C-34000-38) From Value Plastics Inc: Male luer to 200-series barb (cat# MTLL230-1), tee connector with 200-series bard (cat# T230-1), Y connector with 200-series barb (cat# Y230-1), female luer cap (cat# FTLLP-1), male luer plug (cat# LP4-1) female luer coupler (cat# FTLLC-1)

  

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Testing Using Pre-made Controller Box

  

This is currently not an available option.

 

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Building Your Own Valve Controller

 

You can build a computer-based electronic valve controller to allow you to control the opening and closing of valves and pressure in the flow lines of your chip. The controller is comprised of 1) an electronic controller box and 2) a set of electronic valves. The Quake Lab has developed a cheap USB-based valve controller system to drive the microfluidic chips from a computer using LabView or Matlab. This system uses pressurized air and solenoid pneumatic valves to control the valves in the chip. You can use the same pressure regulators and plastic Luer manifolds shown in Testing Using Manifolds to supply the air to the solenoid valves. Injection of fluids into the chip can also be done as shown in this section when using the computer controlled valves.

The following link contains files with parts list information and assembly instructions:

http://microfluidics.lbl.gov/valve-controllers 

 

If you have any further questions regarding the computer-based controller, please contact Rafael Gómez-Sjöberg at rafaelgs#stanford.edu.

  

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Leadership

 

Adam White | Director of Stanford Microfluidics Foundry

Email:  akwhite@stanford.edu

Phone:  604-992-6484

Fax:  (650) 736-1961

 

 

Location and Hours of Operation

 

Location	Hours
Stanford University, Bioengineering Department 318 Campus Drive Clark Center, Room E300 Stanford, CA 94305 USA Lab Phone:  (650) 724-7383 Fax:  (650) 736-1961	9 a.m. - 5 p.m., Monday to Friday   24/7 access for trained users

You can find a map of our location using the Stanford's searchable campus map.

 

 

Links and Resources

 

• Visit our website for more information:  http://www.stanford.edu/group/foundry/index.html

    

 

Publication Acknowledgement

 

As with all Stanford Service Centers, credit must be given to the Stanford Microfluidics Foundry for data that results in a publication. If the work done at Microfluidics Foundry produces data resulting in a figure in a publication, you are required to acknowledge Microfluidics Foundry in the publication. Further, if Microfluidics Foundry staff members provided significant experimental design, data interpretation, or other intellectual contribution (as evaluated by the PI), then it is expected that these individuals will be coauthors on the publication.

 

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Contacts

Name	Role	Phone	Email	Location
Adam White	Director	604-992-6484	akwhite@stanford.edu	Stanford Microfluidics Foundry, 318 Campus Dr., Clark Center, E300, Stanford, CA, 94305