Application Manual for Xtend Microarray Pins

 

Detailed pins specifications can be obtained online at www.labnext.com/xtend.html

 

Precautions

Extra Precautions for 80um and 100um Pins

- Tips are extremely fragile.
- Do not touch tips by hand.
- Do not drop pins.
- Try to minimize lateral forces applied to the pin tip.
- FILTER all liquids used for printing and cleaning the pins.

All other precautions also apply to 80um and 100um pins.

140um through 700um Pins
 
All parts of the Xtend pins are rigid and resistant to chemical, mechanical, and temperature-related wear. 
However, certain precautions must be taken to prevent damage, extend their operating life, and achieve 
the best results when printing microarrays.

The ceramic tip can be broken if it is bent with significant force or the pin is dropped on a hard surface.
When handling and operating pins, treat the tip as if it were made of glass. This will help prevent it from 
breaking. 

- The capillary channel of the pin is very thin. It can become clogged with small particles contained in the printing 
  liquids or in the air. 
- Always use filtered liquids for printing. 
  Example of filters: http://www.daigger.com/store/syringe-filters-30-mm8439y1/18893?section=20
- Store pins in a clean environment.
- Avoid touching tips by hand.
- The capillary channel of the pins can become clogged with salt crystals if printing solution is left in the 
  pins and allowed to dry. 
- Always wash and dry pins after printing either manually or in the microarrayer.
- Do not heat pins above 120 C.

 

Operations

We recommend starting with larger-diameter pins to gain experience before moving to the 
smallest diameters. Larger-diameter pins have wider capillary channels and are less sensitive to 
contaminants such as dust or other particles that can clog the channel. They are also easier to clean.  
An ideal pin size to start with is 0.3mm.

Always make sure that the capillary channel is clean before using the pin. If the capillary channel is clogged, the 
pin will not pick up liquid. To check the pin, use a syringe with a thin dispensing tip. 
Insert the tip into the metal part of the pin and try to blow air through it. If air passes through the pin, 
the capillary channel is clear. If not, the pin requires cleaning before use.

Keep the pin clean after use. If the printing buffer or printing material is left and dried on the surface of
the pin, it may produce inconsistent spots the next time it is used until the residue is dissolved and washed away.
 

Cleaning

For cleaning during normal printing operations, dip the pins into the cleaning solution for 4-5 seconds to 
allow them to pick up the maximum amount of liquid. Dry the pins in a drying station that allows air to pass through 
the inner channel of the pin. Typical cleaning solutions for microarray operations are distilled water or 10% 
alcohol.

For printing with other substances choose cleaning agent that can dissolve such substances.

For cleaning pins with clogged channels use stronger solvents. 90% alcohol or acetone can be used for cleaning 
pins. 

Sometimes, boiling pins in water helps dissolve particles that block the capillary channel. When boiling, 
keep the pin from touching the bottom of the boiling vessel.

Use an ultrasonic cleaner.


If necessary, use syringe with a dispensing tip attached to blow air or washing liquid through the pin to clean
the capillary channel. You can blow air both ways through the pin. See the image below.
	






NOTE: Do not do this with 80um and 100um pins. You will most certainly damage the tip.


Basic Troubleshooting

Visual observation (under a microscope or with a visible-light scanner) may not show features otherwise
detectable at the wavelengths used by fluorescent scanners. Conversely, sometimes visible-light observation shows 
artifacts in printing that will not appear in the scanners or may disappear during microarray processing.

However, for the purposes of this manual, we used a visual (paper) scanner at 2200 px/inch resolution to illustrate
and discuss common undesirable patterns that may occur in microarray printing.
Microarrays were printed on plain glass slides with a water-based blue dye solution.
Spot size was 140um, and the distance between spots was 230um.

Ideally microarray spots should appear uniform after printing. Uniform means equal in shape, size and
even distribution of the printed material across the surface of the pin.





“Doughnut” Spots

“Doughnut” spots result from a splash or a “vortex ring” (or “toroidal vortex”) in the deposited liquid when the
pin hits the surface at high speed. 
The speed at which the pin leaves the microarray substrate is equally important.

More about Vortex Rings -  http://en.wikipedia.org/wiki/Vortex_ring




To produce the doughnut-shaped spots shown in the image above, we intentionally increased the vertical speed
of the microarrayer by approximately 50% over the speed at which it did not produce doughnut spots.
If the liquid is sufficiently viscous to retain this shape for a second or so, it dries and leaves the
spot in the shape of a two-dimensional projection of the ring.

We do not recommend any particular "optimal" speed that would prevent the formation of doughnut spots because 
the process also depends on the physical properties of the microarray buffer, such as viscosity and surface 
tension, and on the hydrophilic properties of the substrate surface.

Reduce Vertical Speed
The first remedy for doughnut-shaped spots is to reduce the vertical speed of the microarrayer as the
microarray pin approaches the surface of the substrate and leaves it.
However some microarray systems ( with stepper motors ) may move in fast increments that are not controllable.
Although the average vertical speed may be low, the movement can consist of a series of fast downward
steps, causing the pin to move too quickly when it hits the surface. For such microarrayers, which use relatively large steps,
controlling vertical speed may not be an option.


Adjust Printing Buffer Composition

Adjusting the physical properties of the printing buffer is another way to address doughnut-shaped spots.
You can slightly increase the viscosity of the liquid (for example, by adding glycerol to water-based buffers). 
Glycerol - http://en.wikipedia.org/wiki/Glycerol
Higher viscosity will reduce mobility of liquid and therefore prevent "splashes" at deposition.

Another approach is to decrease the surface tension of the buffer. This is usually achieved by adding a small amount 
of detergent (SDS, for example, in a concentration of 0.01–0.1%) to the buffer.
SDS - http://en.wikipedia.org/wiki/Sodium_dodecyl_sulfate
Lower surface tension changes spots shape - typically a hemisphere, making it lower ( "flatter" ).  Lower 
surface tension also eases detachment of the droplet from the pin tip when the pin moves up.


Printing with an Unfiltered Solution

The image below is a typical example of printing with a buffer that contains floating particles. 
In order to produce it we did not filter the buffer after dissolving the blue dye in it. A portion of the dry
dye did not dissolve and remained as small floating particles.

Particles can partially block the capillary channel, causing variations in the amount of liquid delivered to the spots.
They also tend to form conglomerates within the spots’ perimeter and often cause spots to change shape and 
even move sideways after deposition.  




In general, random-looking uneven features in the spots indicate that small solid particles are present
in the buffer.
Such a buffer should be filtered using filters described above on this page.  
Example of filters: http://www.daigger.com/store/syringe-filters-30-mm8439y1/18893?section=20

Printing on Non-Uniform Surfaces

Non-uniform surface of a microarray substrate can be a cause for non-uniform spots. Often variations in the surface
properties cannot be observed prior to printing.
The appearance of repeated similar features in a number of spots indicates that the surface of the microarray
substrate may have a defect.  

In order to produce the microarray below, the glass slide was wiped in vertical direction with a tissue soaked
in acetone. Although it left no visible trace, the spots printed on the left-middle part of the surface 
of the microarray feature similar blank areas in the shape of vertical stripe.





Measuring and Controlling Deposition Volume

You cannot explicitly control deposition volume for a pin. You need to use a different pin if you 
want to print bigger or smaller spots.
Moreover, deposition volume may vary for each pin-buffer-surface combination. It is advisable to 
measure deposition volume delivered in each spot in your particular environment.  
(The table provided on www.labnext.com/xtend.html is for the distilled-water/plain-glass 
combination, which may be very different if you are using viscous buffers or absorbing substrates.)
To measure deposition volume, we recommend loading the pin manually with a laboratory pipette (0.1 uL) 
to be sure that all this liquid is inside the pin tip. Then print as many spots as necessary until 
the pin runs out of liquid. Divide 0.1uL by the number of spots printed to obtain a fairly 
accurate estimate of each spot volume.
For a 1nL deposition volume, the pin should produce approximately 1,000 spots from a 1uL load.

There is a way of controlling deposition volume for absorbing surfaces such as porous membranes 
(nitrocellulose). Such a material can absorb a fairly large volume of liquid over a given time interval. 
The longer the pin stays in contact with the surface, the larger the volume absorbed. Thus, regulating
contact time of the pin tip with the substrate gives you the ability to control deposition volume. 
As mentioned above every buffer - surface type combination produces different results. 
Experiments should be conducted to estimate the actual deposition volume and its dependence on the
contact time.

Non-absorbing surfaces, such as some plastics, glass, or metals, do not show a significant 
dependence of spotting volume on contact time.


Dimensions Reference for Xtend Microarray Pins (Version 2.0).




NOTE: Version 2.0 Xtend pins have been available since October 2017. 
V2.0 replaces V1.0 and is fully compatible with all LabNEXT print heads, with equivalent performance in every 
aspect. 
Key dimensions of V1.0 and V2.0 are the same. 
However there are a few minor differences between the two versions.
- V2.0 pins have a slightly longer collar (the upper, wider part).
- V2.0 pins have a different-looking metal surface due to a new manufacturing technology.

Specs for the V1.0 version of Xtend and Xtend RM pins can be found here:
www.labnext.com/documents/images/pins_manual/xtend_v1.png