Oligosaccharides paired with proteins create conjugates that develop into dynamic reagents for high throughput characterization of the recognition capacity of monoclonal antibodies, carbohydrate binding modules, carbohydrate active enzymes and other oligosaccharide binding proteins.
Such microarrays can be readily printed utilizing Arrayjet non-contact printers onto a range of substrates. A well-characterized array library of plant oligosaccharides has been developed by the Department of Plant Biology and Biotechnology at the University of Copenhagen, utilizing robust Arrayjet microarrayers (Pedersen et al., 2012).
Image Credit: Arrayjet Ltd
Experimental design
Sample preparation
Preparation of oligosaccharide samples (1→4)-β-D-mannohexaose and (1→5)-α -L-ara binopentaose was conducted via chemical synthesis or hydrolysis of source polysaccharides succeeded by a conjugation reaction to BSA. Printing buffer containing 55.2% glycerol, 44% water and 0.8% Triton X 100 was used to dissolve the samples.
Substrates
A variety of substrates were selected for oligosaccharide printing, including coated Nexterion® NC, H, P, E, glass slides layered with nitrocellulose (FAST) and nitrocellulose membrane.
Inkjet printing
200 pL spots were printed onto all glass slide types. Each drop dispensed by an Arrayjet microarrayer is 100 pL. 600 pL spots were printed onto the nitrocellulose membrane.
Microarray probing
Subsequent to the blocking process (Pedersen et al., 2012), nitrocellulose membrane and glass slides were incubated with anti-mannan mAbs- LM21, or anti-arabinan mAb LM6 for 2 hours (1/10 antibody dilution) with PBS and PBS containing 0.05% Tween 20, respectively.
All array types were cleaned using PBS. They were then incubated for 2 hours (1/5000 antibody dilution) with anti-mouse or anti-rat secondary antibodies. Utilizing a substrate consisting of 5-bromo-4-chloro- 3-indolylphosphate (BCIP) and nitrobluetetrazolium (NBT) in BCIP/NBT, nitrocellulose microarrays were then developed.
Image acquisition and analysis
Utilizing a flatbed scanner (Cannon 8800, Søborg, Denmark), nitrocellulose membrane arrays were scanned. The slides were scanned with a slide scanner (GenePix 4100, Molecular Devices, Sunnyvale, USA). The output was evaluated with the assistance of software (ImaGene 6.0, BioDiscovery, ElSegundo, CA, USA).
Results
The images displayed in Figures 2A-F were acquired using BSA conjugated to (1→4)-β-D-mannohexaose and (1→5)-α -L-arabinopentaose printed in six repeats across various concentrations ranging from 2 mg/mL to 0.5 μg/mL (Figure 2A-E) and 2 mg/mL to 30.5 ng/ mL (Figure 2F).
The more sensitive detection was seen at 2 μg/mL (for (1→4)-β-D-mannohexaose) with Nexterion®E slide (Figure 2D) and the Fast slide (Figure 2E). The detection exhibiting the least sensitivity was observed at 125 μg/mL (for (1→4)-β-D mannohexaose) with Nexterion®P slide (Figure 2C).
Additionally, arrays printed on the Fast slide (Figure 2E) generated a consistent spot size for all concentrations and were of higher quality in contrast to arrays of Nexterion®E slide.
Nitrocellulose membrane arrays generated a sensitive detection at 122.1 ng/mL (Figure 2F). For reproducibility purposes, 12 copies of arrays probed with mAb LM16 and mAb LM21 were printed onto nitrocellulose membrane and FAST slides.
Example replicate arrays are exhibited in Figure 2I and 2J. The average spot signal from 3 replicate arrays was assessed, and data was extracted.
Data analysis
The data sets from the mean spot signals of arrays (Figure 2I and 2J) were plotted against one another and r2 values were determined. Low variability was observed between the arrays sets with r2 values greater than 0.9 in every case.
Figure 2. Reproducibility of the microarrays was tested by printing 12 copies of arrays on both nitrocellulose membrane (A) and nitrocellulose coated glass Fast slides (B). Representative replicate arrays are shown and also graphs of mean spot signals from 3 arrays plotted against each other. Axes on the graphs are relative mean spot signals. r²= coefficient of determination, equals to the proportion of variability explained by the linear relationship between X and Y.
Image Credit: Arrayjet Ltd
Advantages of Arrayjet microarrayers
Arrayjet non-contact printers release samples by an exceptionally reproducible piezo-actuation process creating high-quality spots that demonstrate consistent reproducibility over long print runs and batch-to-batch.
This offers an advantage over pin-based contact spotters, where a decrease occurs in the array quality across longer print runs due to the unavoidable wear of the pins with repeated usage (Pedersen et al., 2012).
Arrayjet microarrayers offer flexibility in printing a large number of probes on the same slide with a faster speed. This not only improves the throughput but prevents buffer loss through a concomitant concentration of samples throughout longer print runs (Pedersen et al., 2012).
Conclusion
The results of Pedersen et al., 2012 show that Arrayjet microarrayers can effectively print dynamic, high resolution plant oligosaccharides onto a wide range of substrates at high speeds.
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.
- Pedersen, H.L., Fangel, J.U. , McCleary, B., Ruzanski, C., GroRydahl, M.G., Ralet, M-C., Farkas, V., Schantz, L., Marcus, S.E., Andersen, M.C.F., Field, R., Ohlin, M., Knox, J.P., Clausen, M.H., and W.G.T. Willats. (2012). Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research. Journal of Biological Chemistry, 287, 39429-38.
About Arrayjet
Arrayjet provide instruments and services to pharma, diagnostic and lifescience 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. 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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