This article outlines how fluorescent tagged antibodies were successfully printed onto Schott Nexterion® epoxysilane slides utilizing Arrayjet JetStar™ Protein Printing Buffer C to generate high quality spot morphology.
It offers evidence supporting the reproducibility and consistency of antibody arrays fabricated on Arrayjet microarrayers. Printing buffer plays a critical role when developing a protein array experiment.
To accomplish high quality spot morphology, the viscosity of intended printing targets using Arrayjet Inkjet technology should be between 4-20 centipoise (cP). Arrayjet possesses an optimized range of printing buffers to print various sample types.
To start, each printing buffer should be analyzed for each type of protein in order to attain excellent spot morphology without influencing the protein functionality. High quality spots were achieved when printing Immunoglobulin-G (IgG) using JetStar™ Protein Printing Buffer C.
The print head can deliver 100 pL droplets with a maximum delivery volume of 10 nL achieved by delivering a number of drops per spot. One drop generates spots approximately 100 μm diameter in size.
By expanding the number of drops per spot throughout the printing process, the spot diameter can be controlled. However, the spot diameter also depends on factors such as temperature, humidity, the printing buffer and the surface chemistry of the slide.
Experimental design
Sample preparation
Protein sample, antihuman IgG (Fc specific)-Cy3 fluorescent tagged, developed in goat (1mg/mL), (Sigma) was successively diluted 1:2 times to acquire four different concentrations of IgG samples when utilizing Arrayjet’s JetStar™ Protein Printing Buffer C (as displayed in Table 1).
Table 1. Dilution Series of Anti-human IgG-Cy3 samples. Source: Arrayjet Ltd
| Sample number | IgG (µg/mL) |
|---|---|
| 1 | 500 |
| 2 | 250 |
| 3 | 125 |
| 4 | 62.5 |
Inkjet printing
An Arrayjet Sprint microarrayer with JetMosphere environmental control system was used to print arrays onto Schott Nexterion® epoxysilane slides. Ten replicate spots per IgG concentration were printed at 100, 200 and 300 pL delivery volume. The temperature and humidity (RH) were held between the ranges 15-20 °C and 40-60% RH, respectively.
Drying
To ensure high quality spot morphology, the epoxysilane slides were incubated for 1 hour at 37 °C.
Image acquisition
Images were acquired at 532 nm wavelength and PMT gain 160, using the GenePix® 4000B scanner (Molecular Devices) to scan each of the slides.
Data acquisition and analysis
Data acquisition was conducted using the GenePix® Pro 6.0 4000B. Local background corrected values were employed. Average values of the median F 532 of all replicate spots were used for signal calculation.
All features were coordinated with resized diameters. The successful print run was validated by measuring the CV % values for all IgG concentrations with various spot sizes.
Results
Following image acquisition, the results obtained demonstrated high quality spot morphology, as shown in Figure 1. A consistent horizontal pitch was accomplished, emphasizing optimum placement accuracy during the print run.
Figure 1. Image acquisition showing high quality spot morphology. A: 1 drop per spot of Anti-human IgG sample prepared in JetStar™ Protein Printing buffer C. The first two spots indicate 500μg/ml sample concentration following the pattern of 2 adjacent spots for each serial dilution (250μg/mL, 125μg/mL and 62.5μg/mL). 10 such repeats of fluorescent labeled anti-human IgG-Cy3 were printed to form one mini-array. B & C: 2 and 3 drops per spot respectively indicate similar morphology and pattern as explained in A.
Image Credit: Arrayjet Ltd
Figure 2 and Table 2 present the results acquired subsequent to data acquisition and analysis. After local background subtraction, the signal shown was the mean average of the median spot intensity replicates.
Figure 2. Graphical representation of signal intensity versus IgG concentration for different delivery volumes. A linear increase in intensity with successive IgG concentrations for 1, 2 and 3 drops per spot.
Image Credit: Arrayjet Ltd
Table 2. Quantitative data analysis of IgG samples. Source: Arrayjet Ltd
A. Mean spot diameters calculated by averaging all replicates of serial dilutions for each drop per spot.
| Mean spot diameter (µm) | Drops per spot | ||
|---|---|---|---|
| 1 | 2 | 3 | |
| 145.9 | 198.16 | 240 | |
B. Mean signal intensity and CV% values of various IgG dilutions for different 1, 2 and 3 drops per spot.
| IgG concentration (µg/mL) | Drops per spot | |||||
|---|---|---|---|---|---|---|
| 1 | 2 | 3 | ||||
| Mean intensity | Mean CV % | Mean intensity | Mean CV % | Mean intensity | Mean CV % | |
| 500 | 2193.03 | 2.95 | 2379.1 | 1.2 | 2668.63 | 3.33 |
| 250 | 1345.92 | 9.74 | 1489.02 | 9.49 | 1643.81 | 5.95 |
| 125 | 758.7 | 8.5 | 866.78 | 4.62 | 925.78 | 4.44 |
| 62.5 | 430.43 | 3.91 | 482.72 | 4.62 | 512.61 | 5.35 |
There was a linear relationship between protein concentration and signal intensity, as well as showing a consistently low variability between replicates.
Conclusion
High quality spot morphology, reproducibility and placement accuracy were discovered when printing antibodies onto epoxysilane slides utilizing Arrayjet’s JetStar™ Protein Printing Buffer C.
The spot diameter and signal intensity improved with greater IgG delivery volumes illustrating the level of printing flexibility made possible with Arrayjet inkjet Microarrayer.
Arrayjet microarrayers generate high quality spots with exceptional printing consistency in a strong and reliable manner that fulfills customer-specific requirements.
References
- McWilliam, I., Chong Kwan, M. and Hall, D. (2011). Inkjet Printing for the Production of Protein Microarrays. In Korf, U.(ed) Protein Microarrays: Methods and Protocol, Humana Press, New York.
About Arrayjet
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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