The microarray format has emerged as a very powerful tool for the quantification or screening of DNA or protein samples. One of the key bottlenecks in obtaining high quality and reliable data is the design and manufacture of the immobilized arrays. Whether working with DNA or protein microarrays, the selection of substrate and subsequent assay development are very critical, but without a reliable array printing instrument, the quality of data is limited. The use of complex and sensitive instrumentation is required to produce arrays containing small enough features (spot or dot) with good reproducibility and accuracy. Non-contact type printing is an optimal tool to obtain these high quality chips. Although there are only a few options for this type of instrumentation, I will address the Gesim Nanoplotter 2.0E. These are complex instruments and the evaluation could be very long when describing them in-depth, so I will limit this evaluation to the general benefits of non-contact printing and how well the Gesim performs those tasks as well as the general usefulness and reliability of the machine. I have extensive experience with this instrument, but limited in scope to mainly protein microarrays printed on thin film nitrocellulose slides.
The Nanoplotter 2.0E is considered the high throughput machine of the Gesim Nanoplotter range. This is due to the larger platen, which will hold close to 100 1x3 inch slides and has high printing density, a claimed 2500 dots per cm. The Nanoploter 2.0 has a smaller platen with a capacity of 60 slides and the same high printing density. The Nanoplotter 1.2 platen holds only 40 slides and offers a lower maximum printing density of 1200 spots per cm.
Low spot to spot variation, adjustable spot size, limited sample contact, very small sample volume usage and no contact between the substrate and the printing tip are general advantages to a non-contact printer. Of course the drawback of the non-contact printer is the complexity of the instrument and cost. After extensive printing experimentation, the Nanoplotter 2.0E dependably yields spot-to-spot percentage coefficient of variation (%CV) of less than 6%. This is fairly good judging by the industrial standards and usually the Nanoplotter 2.0E will deliver spot-to-spot %CV’s in the 2 to 4% range.
Regarding the adjustable spot size, the Nanoplotter 2.0E is capable of printing a range of sizes, but the adjustment is not simple. It requires optimization of the dispensing voltage in each individual tip and is a delicate adjustment. We generally found that each tip dispenses optimally at a certain voltage at which the spot consistency is very reproducible and missed spots are minimized. Deviating from the optimal setting to change spot size increased the amount of missed spots. However, spot sizes can still be adjusted by changing the number of droplets to be dispensed on one feature. The Gesim instruments can use two types of tips, the Nanoliter Pipette tips and Picoliter Pipette tips. The former will dispense droplet sizes of approximately 0.4 nL and the later will pipet droplet sized to 100 pL. We have evaluated both tip types with varying results. The Nanoliter Pipette tips yield spots slightly larger than 220 um, which is typical for the industrial standard. This tip at this spot size has yielded by far the best results in regards to consistency of spot size and lack of in-run printing errors. We had much difficulty obtaining good results with the Picoliter Pipette tips. The spot sizes were impressively small, in the 150 um range, however, spot-to-spot variability, alignment and in-run errors prevented extensive production use with these tips. The tip will draw about 2 – 3 µL of sample and can reliably print over 2000 spots with one pick-up. It is possible to print over 4000 spots with one pick-up but the last third will taper off in intensity when printing so many.
The Nanoplotters can hold up to 8 of these tips, enabling the instrument to have a very high printing capacity and complete print jobs quickly. We started our evaluations with 4 tips with good results and were able to print large numbers of slides quickly. However, we also found some tip to tip variability which would lead to well to well inconsistencies. The finest slides we were able to print utilized a single tip format, which increased the print job time, but yielded extremely low spot-to-spot %CV’s. Creating matched sets of tips is expensive but ideal for fast, reproducible printing. Spot size of individual tips may vary over the tips lifetime, so single tip printing always yielded the absolute best level of performance.
The instrument itself is built of high quality materials and is designed and constructed well. Although an expensive and complicated machine, set-up was straightforward, although familiarity with instrumentation is of great value when assembling the machine. The initial set-up is performed with a Gesim representative but routine maintenance requires some general instrumentation familiarity and mechanical aptitude. The footprint of the instrument is reasonable and the arrayer itself has a robust enclosed chamber. The arrayer footprint is about 4 feet by 3 feet, along with computer terminal, dispensing pumps and some wash and rinse fluid reservoir bottles.
The array layouts or designs the instrument is capable of producing are fully open and much less restrictive than any printer I’ve worked with. Array layouts of any number, patterns and locations are possible. This open format printing is enabled by complex software, which takes extensive use to master. The user interface is straightforward and intuitive, but there are a large number of parameters and settings to implement when creating a print protocol. Fortunately, the software is fairly easily navigated and users will find protocols easy to establish after some familiarization. The software overall is powerful with additional options enabling various source plate set-ups, simulated runs to check your protocol accuracy, missed spot repair and multiple print modes.
Instrument maintenance is surprisingly small for a machine performing such exacting maneuvers. We have had some incidents of the instrument failing, not due to operator error, but customer support was fairly quick. A US representative of Gesim or for larger issues, a German Gesim representative responded quickly to inquiries and addressed problems for the most part. One major instrument failure was not addressed as quickly as expected, resulting in several weeks downtime. The main service drawback occurred when new or replacement parts were required. Delays were experienced due to communication and shipping times from European sources. Customer service for questions or issues regarding general instrument operation is spotty. Obtaining information for operating guidelines was not readily available. We were provided with a two day instructional visit from a US Gesim representative, but due to the large amount of information required to run the instrument, the training was introductory. Additional information regarding operating procedures was provided by Gesim after inquiries; most of it useful, but not all. Unfortunately, there is generally little information available regarding the operation of instruments such as these. Companies or groups who have mastered printing with these types of instruments usually have done so at large costs to themselves and jealously guard their methods as proprietary process information and thus, it is difficult to come by. Routine maintenance includes cleaning the tips, managing the flushing solutions and washing stations and flushing the system to clean.
Gesim offers many accessories with the Nanoplotters. With our system, we obtained a stroboscope for optical droplet detection, removable slide platen, humidifier and Z-Level sensor. With the exception of the removable platen, all these accessories I feel are invaluable to maintain a reliable printing system.
Overall, I feel this is a fairly good instrument which produces arrays as good or in most cases better than anything else on the market. The drawback is that this was a very expensive instrument. Additionally, it performs best when it is operated and maintained by a single technician, making it a possible strain on resources. The maintenance issues are very concerning when using them in a manufacturing role. If this instrument were slightly more reasonably priced and were slightly more reliable I would rate it as excellent. More robust and less expensive machines for array printing are available, however, unfortunately, they cannot come close to the level of performance the Nanoplotter provides.
Scientist
Research and Development
Gentel Biosciences