This article highlights evidence of feature reproducibility and consistency across slide batches printed when utilizing Arrayjet Marathon microarrayers. It details the successful batch-to-batch printing of fluorescent tagged Bovine Serum Albumin (BSA) onto Schott Nexterion® epoxysilane slides to generate high quality spots.
Experimental design
Sample preparation
Bovine Serum Albumin (BSA-IgG free, 1mg/mL, Sigma) was diluted in a 47% glycerol buffer including 1 μg/mL Rhodamine B. Deposition of the sample volume of 20 μL across 36 wells of a 384 well microplate.
Substrates
Arrays were printed onto Schott Nexterion® Epoxysilane slides.
Inkjet printing
The Arrayjet Marathon microarrayer dispensed drops 100 pL in size. One drop per spot was printed per sample, in triplicate across 48 mini-arrays.
Three sample redraws were carried out with 1.3 μl of sample aspirated each time in order to print a batch of 100 slides. The temperature and humidity (RH) were held between the ranges 15-20 °C and 40-60% RH, respectively.
Image acquisition and analysis
Slides at various tray positions were scanned utilizing GenePix® 4000B scanner (Molecular Devices). Images acquisition was performed under a wavelength of 532 nm with 100% power gain and a PMT of 230.
Data analysis
A GenePix® Pro 6.0 4000B was used for data acquisition. Mean intensity values of the median F 532 (minus background) of all replicate spots were utilized for calculating the signal. The average diameter of all features was documented.
Results
Intra batch analysis
The Arrayjet Marathon microarrayer’s printing reproducibility was demonstrated within one batch of 100 slides. An excellent spot morphology was seen across all slides with consistent spot positioning (Figure 1).
Figure 1. Representative image showing 3 replicates of BSA sample across 4 miniarrays. Good spot morphology and consistent spot positioning can be observed across all arrays.
Image Credit: Arrayjet Ltd
It was observed that the intraslide %CV values (Table 1) were low, thereby signaling minimum variability within a single slide.
The average intrabatch CV value was reasoned to be 4.14%, illustrating consistent signal intensity from every spot printed within a 100 slide batch. The spot size across the entire batch was in range of 110 μm to 115 μm.
Inter batch analysis
To effectively illustrate inter batch printing consistency over time, two batches of 100 slides were printed on the same day and then again over two separate days. Table 1 shows the results following data acquisition and analysis.
Table 1. Intra batch and inter batch slide analysis. Arrayjet Marathon microarrayer demonstrating printing reproducibility and spot size consistency. (Mean intensity = median 532-background 532). Source: Arrayjet Ltd
| . | ||||||||
|---|---|---|---|---|---|---|---|---|
| Slide position | ||||||||
| 25 | 50 | 75 | 100 | Mean intensity | Mean standard deviation (SD) | Mean CV (%) | Mean diameter (µm) | |
| Intra-slide CV | ||||||||
| Batch 1 | 5.8 | 5.34 | 6.13 | 5.67 | ||||
| Intra-slide diameter (µm) | ||||||||
| Batch 1 | 113.01 | 110.89 | 115.97 | 112.76 | ||||
| Intra-batch signal intensity (MFU) | ||||||||
| Batch 1 | 20509.18 | 21693.65 | 19704.73 | 21184.5 | 20773.02 | 861.72 | 4.14 | 113.15 |
| Inter-batch analysis (2 days) | ||||||||
| Batch 1 | 20509.18 | 21693.65 | 19704.73 | 21184.5 | 20451.71 | 892.72 | 4.36 | 113.15 |
| Batch 2 | 19469.52 | 21150.03 | 19249.51 | 20652.58 | 108.81 | |||
| Inter-batch analysis (1 day) | ||||||||
| Batch 1 | 17994.8 | 20454.08 | 18874.19 | 20042.05 | 19175.98 | 974.92 | 5.08 | 111.37 |
| Batch 2 | 17679.36 | 19192.64 | 19262.74 | 19907.98 | 111.71 | |||
The average signal intensity acquired from two individual batches over two different days demonstrated a low %CV value of 4.36%, signifying consistency in print quality and inter batch reproducibility over time.
The mean CV value for printing conducted on the same day was 5.08% indicating printing reproducibility between successive batch runs. The spot size across all of the batches was in the range of 108 μm to 113 μm.
Print head nozzle performance
Arrayjet printing technology utilizes the Xaar™ print head for sample deposition onto substrates. A low volume 12 JetSpyder docks with the print head to load 12 samples synchronously into various nozzle sets of the print head (Figures 2A and B).
Figure 2A and B. Arrayjet print head and JetSpyder™ operation. The JetSpyder™ is docked to the print head, and moved to the wells of the microplate containing samples to be arrayed. 12 samples are simultaneously drawn through the JetSpyder™ into the nozzles of the print head. The nozzles of the print head contain the samples to be printed. As the print head travels in a non-contact fashion across the slide, the samples are deposited in the form of spots on the slide.
Image Credit: Arrayjet Ltd
Analysis of individual nozzle performance was conducted by calculating the average %CV of all spots printed with the same nozzle number (Figure 3). The mean %CV values from the various nozzles were found to be in the range of 3-4% emphasizing both the precision of the spots and printing accuracy utilizing Arrayjet microarrayers.
Figure 3. Individual nozzle performance for spots printed. The individual nozzle performance was analyzed by calculating the mean coefficient of variability (%CV) for these positions. The CV values were calculated to be 4% or lower indicating highly precise and consistent individual nozzle performance.
Image Credit: Arrayjet Ltd
Conclusion
Results acquired following intra batch and inter batch slide analysis demonstrate excellent printing reproducibility (~5% CV values) and spot size consistency when employing Arrayjet Marathon microarrayers.
High quality reproducible spots were generated with minimum spot to spot, slide to slide and batch to batch variability when a protein sample such as BSA was printed onto Schott Nexterion® epoxysilane slides.
Furthermore, the precision performance of the print head’s nozzle combined with rapid in-situ printing makes Arrayjet non-contact technology the ideal choice for manufacturing many slide batches over a certain period of time.
References
- McWilliam, I., Chong Kwan, M., and Hall, D. (2011).Inkjet Printing for the Production of Protein Microarrays. In: Protein Microarrays: Methods and Protocols. (U. Korf, ed) Humana Press, New York.
About Arrayjet Ltd
Arrayjet provide instruments and services to the pharma, diagnostic and life science industries. Our products use inkjet technology for precision picolitre liquid handling. Arrayjet focus on printing samples to create tools for genomic and proteomic screening, patient stratification and clinical diagnosis.
The proprietary printing technology is fully automated and delivers benefits of ease of use, precision, reproducibility, efficiency of manufacture, and total process-control.
Arrayjet’s patented technology simultaneously aspirates and prints multiple samples on-the-fly. This is a proven platform and its non-contact bioprinting is ideal for microarray and 96 well microplate manufacture; as well as bioprinting onto biosensors, biochips, MEMS devices, microfluidic devices, membrane sheets and into nanowell applications. Most substrates are compatible with the technology.
Arrayjet instruments offer the largest manufacturing batch size of up to 1000 slides, allowing over 18,400 samples to be loaded at once. The instruments are modular and scalable, enabling customers to increase capacity as their requirements grow. They combine the fastest and most reliable instrumentation on the market with the versatility to print any biological sample type onto any solid substrate.
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