Advanced Microarrayer System for Making DNA Micro-Arrays

Ratnesh Singh Sengar, A.K. Upadhyay, K.D. Lagoo, R.K. Puri, Manjit Singh Division of Remote Handling and Robotics, Bhabha Atomic Research Centre, Mumbai, India AbstractThis paper discusses the advanced technology of control and automation for printing DNA microarrays. With greater automation of facilities, the motor and control system have been compelled to improve the speed and precision of machine tools and quality control machines. Recently, the linear servo motor technology underwent to the growing demands for optimized productivity to reduce operating overheads, high stiffness, reduced maintenance requirements, longer product life and enhances system performance. Based upon this, The Advanced Microarrayer is a very precise computer controlled 3-axis robotics system to deposit high-density, gridded arrays of cDNA, genomic DNA or similar biological material on microscope slides. It promises to make mutation detection and gene expression analysis less time consuming and more efficient, thus revolutionizing areas of biotechnology and medicine. The performance, accuracy and repeatability of the system are evaluated by printing test patterns and quantifying various spot parameters using statistical methods. Keywords: DNA microarrays, advanced microarrayer, linear motor, stiffness, control and automation,
spotting software, state-transition diagram, data analysis.

1. INTRODUCTION DNA microarray technology is an experimental approach to study all or a large number of genes in a given organism simultaneously. It is capable of depositing thousands of extremely small droplets of genetic material on a small surface area, one droplet at a time, with each droplet containing a different gene. Such a capability permits gene expression experiments using tiny samples of genetic material while obtaining simultaneous data on thousands of genes. The emerging field of genomics promises makes mutation detection and gene expression analysis less time consuming and more efficient, thus revolutionizing the areas of health and medicine. The basic idea is to print the DNA templates (also called target or probe spots) for all possible sequences that can be expressed in a given organism in a two-dimensional array at a high-density on a solid surface. The number of probes can be as many as a few thousands. These targets are then hybridized with fluorescently labeled complimentary sequences in a test sample (DNA or RNA). The fluorescence intensity in each spot of the array is measured through a suitable scanner. The data thus generated includes the location and intensity of the fluorescent signal. The location of the signal identifies the probe/gene being examined while the intensity gives an estimate of the quantity of that particular DNA fragment in the sample [M. Dufva, 2005]. The accuracy demands are high because of the microscopic size of the spots. Various design considerations and the quality parameters to be assessed while making microarrays are also discussed. 2. DESCRIPTION 2.1 Design Features Many advanced features have been incorporated in the design of system taking into account experience of the prototype microarrayer system [Ratnesh Singh Sengar, et al, 2004].

The important considerations while designing a precise 3-axis robot are higher speed, micron level accuracy (at a scale of 30-50cm) and minimum vibration. We achieved this by using a well-damped table; Linear Servo motors with high-resolution encoders. The robot is designed to automatically collect samples from the wells of either a 96 or a 384-well microtitre plate with up to 32 especially made pins and deposits approximately 1 nanoliter on each glass slide. The rigid glass slides are used that is easy to work with than membranes [Maskos U, 1992]. The system can hold four microtitre plates (of either 96 or 384 wells) and generate a maximum of 100 standard microscope slides per batch. However, the software can be configured to print a lesser number of slides. The whole system rests on an anti-vibration table and is housed inside a transparent, dust free enclosure. A dehumidifier attach to the enclosure controls its humidity and temperature. 2.2 Hardware Description The microarrayer consists of a three-axis robot mounted on a vibration isolation workstation table as shown in fig. 1.

Fig. 1: Advance Microarrayer System

This table provides a very sturdy support structure with good vibration damping properties by using four shock

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absorbers on the two side support fames. The frame provides a self-supporting structure for the entire apparatus while maintaining isolation between ancillary equipment and the tabletop. Additional components can be added to provide support for an enclosure above the table should this prove necessary. The entire assembly is precisely leveled, while the frame structure below the table is utilized to hold the electronics control panel. 2.3 Components Printing Tip Assembly Printing tip is de1veloped by using participating harden stainless steel (17/4PH) and consists of cylindrical shaped pin of 2 mm diameter with a conical tip as shown in fig. 2. A slot of 70m width and 4mm depth is cut into this conical end by wire cut electrical discharge machining method. The assembly can work with any number of pins up to 32.

Dehumidifier A dehumidifier attached to the enclosure controls its humidity and temperature. Control of humidity is critical for printing as it does not only reduce the evaporation of DNA solutions from the well-plates but also improves signal to noise ratio. The humidity is maintained 45% to 60% in our experiment. 2.4 Linear Motors and Control System Linear servomotors are used for the X and Y-axis while a rotary servomotor with electric cylinder is used for Zaxis to make the system secure. It consists of 300Watt motor for the X and Y axis with a rated speed of 0.3 m/s with 1 micron positional accuracy and 329 N continuous force and a 553Watt Servo motor for the Zaxis fitted with Electric Cylinder, rated at 5800 RPM and 1.13Nm torque. Computer based control system block diagram is shown in fig. 3. Rotatory Servomotor incorporate, through 4X phase detection, can yield an electrical resolution of 8192 counts per motor revolution. This corresponds to a linear resolution of +/- 0.1 microns for X & Y axis and +/- 0.125 microns for the Z-axis. With brush less linear servo motor technology, and the supporting electronics to drive them, the limitations of step motor, rotatory motor & brushed linear motor have been eliminated [Han Ding, Zhenhua Xiong, 2006]. High speed, high precision, fast response, stiffness, zero backlash and maintenance free operation are some benefits of linear servo motors. 2.5 Electric Cylinder A servomotor along with electric cylinder is used in Zaxis to prevent it from free fall and causing damage to the DNA samples glass slides at the time of power failure. The Microarrayer system is a precisely pick and place robot on which most frequent motion in along Zaxis. So, motion mechanism used for Z-axis should be rugged and have longer life. This can be achieved with the use of servomotor in integration with an acme lead screw based electric cylinder. Its robust design, which includes angular contact thrust bearings and roller bearing anti-rotation rod bearing carriage, ensures durability in microarray printing application.

Fig. 2: Printing Tip

When the tip dips into DNA solutions, DNA solution gets collected in the slot through capillary action. When the pin touches a glass slide, it delivers a small amount of DNA solution on the glass slide in the form of an approximate circular spot. Titer Well-Plate The system can hold four standard microtiter plates of 96, 384 or 1536 wells. The well-plate is held by means of a vacuum gripping arrangement. Glass Slides The microscope slides can also be placed anywhere on the work surface. The user aligns the edges of the 1 by 3 inch glass slides to the center of the whole pattern on the tabletop. A group of contiguous slides can be mounted in a column and its location defined by entering the location of the first slide and the number of slides in the group. All slides are held by means of a vacuum gripping arrangement. Cleaning /Drying An ultrasonic chamber (33 KHz) is used for removing the excess solution from the printing tips by ddH2O, followed by drying with a vacuum induced air flow.

Fig. 3: Computer Based Control System for Advanced Microarrayer System

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2.7 Servo Drive The servo drives are used to drive linear servomotor by providing three phase power supply by the use of PWM circuitry. The servo drive consist of a DSP processor which drives a D/A converter to produce an analog torque demand signal by comparing the feedback signal with the command input signal[T.N. Chang, et al., 2006]. This analog torque demand signal drives the servomotor with the desired torque. The servo drive also receives command input of position from the controller and provides the necessary position feedback information to the controller. The servo drive is configured by RS232C serial communication through the host computer. Ultrasonic cleaner and Dryer are interfaced with Solid State Relay and control through the digital output of DAQ device. A realistic dynamic model of the System is constructed [Z. Z. Liu, et al]; various control loop compensator is used to achieve the fast response and less oscillation in the system. The position and speed loop compensators are implemented as software based lead, lag compensator and notch filters. Depending on the bandwidth requirements, different loops may cycle at different rates and tuned for stable system. Current Loop For the current loop we have achieved a bandwidth of 1500 Hz with proper setting of poles and zeros of the compensator. Speed Loop For the speed loop the corresponding bandwidth achieved is 100 Hz. This bandwidth is achieved by a compensator with a pole at origin, an early zero at 50rad/sec. and pole at 500 rad/sec. The pole at origin helps to eliminate steady state offset. The zero improves the phase margin at cross-over and the pole helps attenuate the second resonant peak. Position Loop The main function of the position loop is to position the printing head at defined X-Y coordinates in minimum time with minimum overshoots and settling times. We have achieved the position update rate 40 Hz by adjusting the position damping ratio and forward gains. The transfer function for the position loop is

The corresponding bode plot for the above transfer function and the gain margin and phase margin for the system is indicated in fig. 4.

Fig. 4: Bode Plot of Position Loop

2.8 Software To implement a real-time control as well as userfriendly software, Windows XP with high speed quad core processor is selected. Although Windows XP is not real time operating system, using the high performance high precision hardware timer in the multithreading & multi-core environment make real time control possible on high speed computer. Software has been developed using Microsoft Visual C++, since it provides object oriented programming environment and rich set of functions to implement high performance multithreading applications with graphical user interface on windows platform. Different software resources are discussed here and relationship among them is shown in fig.5.

Fig. 5: Relationship among Different Software & Hardware Resources

Communication Software Communication Software is a 32-bit automation server based upon distributed component object model (DCOM).It facilitates communication between Spotting software and underlying hardware interfaces, e.g. it manages the asynchronous communications between serial port and Spotting software. It also collects data in specially designed FIFO buffer from system through data acquisition (DAQ) device continuously and transfers them to Spotting software. The specially designed first-in first out (FIFO) buffer dynamically

p( s) 500(0.25s + 1) = u ( s) s(0.008s + 1)

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allocates the memory in chunks in advance to improve the performance while normal FIFO buffer allocates the memory each time for each incoming data and thus increases the overload. Spotting Software Spotting Software plays a center role for automating whole system and responsible for controlling everything including printing operation, system calibration, I/O operations, print head cleaning and user interface. Fig. 6 & Fig.7 shows screenshots of running Spotting software. User may configure a printing operation, start, pause, resume and abort it through menu and buttons on user interface. User may also be able to take control of each resource independently. There is a hot key provided for emergency stop so that user can abort the operation at any moment of time. There is also a separate thread running in background which continuously checks status of system and can stop (pause) the operation and can report it to the user if any malfunction is detected in the system. User interface also consist of an indicator to show the progress of operation as the number of dots per slide and total number of iterations. All the information and error, if any, are logged in a log file.

systems behavior during printing cycle in comparison of traditional flow-charts and thus also results in easy implementation of pause/resume behavior and errorhandling.

Fig. 8: State Transition Diagram for Printing cycle. States Contains sub-States and Sub-States of Spotting State are also Depicted where S1 Stands for DetermineSlide SubState, S2 for Determinedotlocation, S3 forPrintDot and S4 for DetermineReplica Andvalidate Sub-State.

The other distinct features of software includes incorporation of password security feature, online calibration of all the resources through main user interface, provision of separate configuration data file for each user, option of generation of an pre-formatted template input file so that user can easily make the complete input file, option of generation of output file as comma separated values (CSV) format so that user can open it in MS-Excel, and even option of selecting solution locations within a well-plate. Imaging Software It is very important for the user to know how well the system is doing its job. Hence any problem needs to be identified and resolved immediately. Imaging software is responsible for grabbing and analyzing images coming from the camera. A PCI based frame grabber card is used for this purpose. Through imaging software, user can see how correctly printing tips are printing the spots on the slides. This software is separate application and doesnt inhibit the normal printing process any way. 3. EXPERIMENT RESULTS AND DATA ANALYSIS In a microarrayer experiment various factors like the physico-chemical conditions of the solutions being spotted, the properties of the printing surface and the inherent properties of the spotting robot affect the quality of the final arrays. Collectively these variations result in a spot whose size, shape as well as its locations is not within an acceptable tolerance or in rare cases leading to the absence of a spot. Detection of these failures at the analysis stage allows for a corrective action at a later stage of the experiment [N. Ramakrishnan et al., 2001-04]. The possible types of error

Fig. 6: User Interface of Spotting Software

Fig. 7: Well-Plate Configuration Dialogue

Printing cycle is implemented in separate thread and conceptualized as finite state machine. It depicted in fig. 9 in a simplified version of state transition graph. Finite state machine concept results better understanding of

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in the process of spotting which are treated as random variables are spot size, amount of probe pipetted by the robot, location of the spot on the slide and the presence or absence of the spot (a discrete variable). 3.1 Statistical Methods used for Error Estimationnormal Distribution We assume the diameter of the spot and the amount of solution (probe) pipetted, like many types of physical measurements, to follow a normal distribution. Moreover this distribution is more appropriate for large sample sizes, as in case of microarrays. x is normally distributed with mean and variance 2, if it has the probability density F(x) = (22 )-1/2 exp{-(x-)2 /( 22)}, - < x < + (1) In case of size of spot, x is the deviation from the mean size. For volume spotted, x is the deviation from the standard amount. For location of the spot, x is the distance from the centre of desired location to the centre of actual location. 3.2 Binomial Distribution The event of the spot being present follows a Binomial Distribution. Let p be the probability of the spot being presented so 1-p is the probability that the spot is not present. 3.3 Error Analysis Solutions of acridine orange and xylene cyanol dyes (two commonly used dyes for staining DNA) were prepared in 3X SSC buffer and were spotted on clean glass slides as well as on photo quality paper. The slides were imaged under a normal/fluorescence microscope that has a CCD camera attached. Alternately, the photopaper was scanned on a flatbed scanner at 1200 dpi resolution to obtain a bit map image of the spot pattern. Image analysis of the spots was done using the image processing software Image Pro Plus version 4.1. Various parameters such as area, perimeter, mean diameter, coordinates of the centre, etc. of each spot were measured using this software. The data from a large number of spots was exported Excel and was analysed further using suitable statistical methods. A typical spot pattern generated with two pins is shown in
A . S p o t s iz e d is t r ib u t io n ( m ic r o n s ) 25 20 Obs erv ed Th e o r itic a l

fig.9. The spot pitch has been kept at 500 microns in all these cases.

Fig. 9: Spot pattern made by advanced microarrayer system

All the following parameters were estimated for our system and will be used for defining the confidence limits while interpreting the results from the Microarray experiments. 3.4 Size Spot The spotting pins resulted in a spot diameter of 100 5 microns. The observed distribution of the spot diameter as compared to a theoretical distribution having same mean and variance is shown in fig. 10. 3.5 Amount of Probe Pipetted The Integrated Optical Density (IOD), which gives the sum of the grey levels of all the pixels in a spot, will be proportional to the amount of probe in the spot. Hence we measured the IODs of all the spots and calculated the deviation from the mean IOD (fig. 10(B)). From these IOD values, we calculated the coefficient of variation (CV) and obtained a CV of ~10% on our system. Typical spotted volumes are likely to be of the order of nano-litre for the quill type pins and CV values between 10 to 20% generally expected. Using suitable references for volume and dye concentration, the volume measurements can be calibrated. 3.6 Location of the Spot We measured the X and Y co-ordinates of the centre of each spot and calculated the deviations from the expected positions. Fig. 10(C) shows the distribution of observed deviation in the spot location and also a normal distribution having same mean and variance. The measured deviation in the spot location is about 5 microns from the expected locations.
C . De v ia t io n in s p o t p o s it io n s ( m ic r o n s ) 40

B. Dis t r ib u t io n o f v o lu m e s p o t t e d

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Fig. 10: Error Analysis of Different Spot Parameters

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3.7 Presence/Absence of a Spot During the limited number of runs, we did not find any spot missing. However, on extensive usage a quantitative estimate of this parameter can be made from a large number of runs of the system and comparison of spot occurrence pattern across runs. This error could result from a partially or fully clogged pin of the spotting robot that might need cleaning or replacement. 4. CONCLUSION We have developed high quality advanced microarrayer system, well known for its successful application of automation in high quality laboratory equipment and life science product. The system hardware consist of the 3-axes robotic motion is highly precise and has a positioning accuracy of the order of microns as measured from the axis movement as well as from the spot locations, and advanced control system scheme. Efforts are on to improve the speed, accuracy and space utilization of the arraying system besides producing better quality spotting pins using linear servo motors technology and advanced feedback technology. In addition to presenting multithreading/multi-core environment for printing process automation, the software part of the system is designed in such a manner that user can control the whole system and configure it with full flexibility. They also get the benefits of several distinguishable features such as online calibration of all the resources and automatic program abortion due to any malfunctions in the system. The hardware and software both are designed to provide high performance automation together with the flexibility required for individual sample handling and custom designed. The system is highly precise and is fail safe. It is capable of rapidly moving to any x-y location on the table within 1 second. Its x-y position can be repeated to an accuracy of +/- 1 micron, for any type of move. It is possible to create 75 slides with 6400

clones each in under 11 hours. Error analysis of the spotting process is very critical for producing consistent and good quality microarrays and will help in minimizing erroneous results in further processes like hybridization and scanning. Various spot parameters that reflect the accuracy of spotting were analyzed. Comparing this system with other micoarrayer system, the automated system is unique in several aspects; this is fully automated system using linear motors technology. All the resources are controlled by single industrial computer and better space utilization. Experimental results indicate that our advanced technology used for control and automation scheme exhibit satisfactorily performance. ACKNOWLEDGMENT The authors would like to thank Dr S.K. Apte and Dr. N. Jawali, Molecular Biology Division (MBD), B.A.R.C., Mumbai for assigning to do challenging advanced microarrayer project. REFERENCES
M. Dufva (Dec. 2005). Fabrication of high quality microarrays. Biomolecular Eng., vol. 22, no. 5-6, pp. 173184. Ratnesh Singh Sengar, et al (2004).Development of a 3-axes robotic system for making DNA Microarrays. International conference on Trends in Industrial Measurement and Automation, Chennai, India, pp. 3339. Maskos U (1992 ).Southern EM. Oligonucleotide hybridization on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotide synthesized in situ. Nucleic Acids Res, vol.20,pp.1679-84. N. Ramakrishnan et al.( 2001-2004).A Microarray experiment management system. NSG Project report, EIA-010366. T.N. Chang, B.Cheng, and P. Sriwilaijaroen (Oct.2006). Motion control firmware for high speed robotics systems. IEEE Trans. Ind. Electron., vol.53, no. 5, pp. 1713-1722. Han Ding; Zhenhua Xiong (Dec. 2006 ). Motion stages for electronic packaging design and control. Robotics & Automation Magazine, IEEE, vol.13,no. 4,pp5161. Z.Z. Liu, F.L. Luo, and M. A. Rahman (Oct. 2005).Robust and precision motion control system of linear-motor direct drive for high speed X-Y table positioning mechanism. IEEE Trans. Ind. Electron., vol. 52, no. 5, pp. 13571363.