Nano-PlotterTMMicroarraying and Picolitre Pipetting

MICROARRAYING

MICROARRAYING

Microarraying and Picolitre Pipetting

Non-contact spotting of picolitre droplets ensures utmost

flexibility and quality for the fabrication of complex

biochips. For many years, the GeSiM Nano-Plotter has been

a reliable companion for both research and production.

The GeSiM Nano-PlotterTM is not just another

microarray spotter. Modular hardware and

an open software guarantee high flexibility

to tackle even the most demanding print job.

GeSiM’s own piezo dispensers handle a wide

range of liquids, not only protein and DNA,

and eject droplets as small as 50 picolitres.

Microscope-based image processing makes

printing onto microelectrodes or other tiny

elements easy. Customised Nano-PlotterTM

versions, e.g. for integration into automated

production lines, are available on request.

A few applications

� Microarrays for research, diagnostics and

drug discovery (DNA, proteins, carbohy-

drates, lipids etc.)

� Spotting of beads and living cells

� Spotting into 96-well plates and onto

many other substrates

� Pipetting of miniaturized assays

� Coating of biosensors, (based on e.g. mi-

croelectrodes), aided by automatic image

processing

Key Features

| “Drop on demand” ensures arbitrary spot layout (including

unlimited replicates)

| No scratches on sensitive surfaces (slides, membranes,

microtitre plates, sensor chips…) via non-contact technology

| Homogeneous spots

| Spot volumes can be changed by varying the number of

drops per spot

| No need to adjust samples to pipettes: either vary pipetting

parameters or choose from different dispenser types

| “Spotting on the fly” for ultimate speed

| Dew point pipetting by chilling and humidification

| Alternative dispensers available for large volumes or viscous

media

|Countless accessories, e.g. automatic target recognition and

post-spotting array repair via microscope camera

www.gesim.de

GESIM Nano-PlotterTM

Configuration

The Nano-PlotterTM comes in two different

sizes with the following common features:

� Small footprint

� Dust cover included

� Z-sensor for height profile measurement

of targets

� 1 — 16 independent piezoelectric pipetting

tips with full fluidic control

� Different tip layouts on the pipetting head

(pitch in parentheses):

1 x 8 (4.5 mm), 1 x 6 (9 mm),

2 x 8 (9 and 4.5 mm), custom tools, etc.

NP2.1: Small platform

for max. 55 slides

NP2.1/E: Large platform

for max. 120 slides

Technical Data Nano-PlotterTM NP2.1 Nano-PlotterTM NP2.1/E

Dispensing area / slide tray

(width x depth, outer dimensions)

302 mm x 400 mm 645 mm x 400 mm

Traversing range in XY-direction (width x depth) 427 mm x 341 mm 777 mm x 341 mm

Footprint main unit 623 mm x 509 mm 973 mm x 509 mm

Height (incl. cover) 375 mm 375 mm

Weight (incl. cover) 30 kg 50 kg

Max. pipetting height 50 mm

Pipetting channels 1 ... 16

Power supply 110...240 V, max. 200 W

� Sample aspiration from 96-well or 384-

well microtitre plates (MTPs)

� XY repetition accuracy much better than

± 10 µm, encoder-controlled, step width

2 µm

� Travel speed up to 500 mm/s

� Function test in stroboscope of each

pipette tip before/after spotting

� Removable target tray

� Array density up to 3000/cm2

� Extra fast travelling mode and spotting on

the fly (spotting while travelling)

Work decks, cooling and humidification

The area inside the hood is variable. Different

removable slide trays (also tailor-made) and

trays for MTPs are available so that one can

trade in slide capacity for source MTPs.

NP2.1/E: Configurations for 5 x 23 slides with one

sample plate (top) and 3 x 23 slides with four sample

plates (bottom). More layouts are available.

Slide trays have handles and lock into place.

An additional tray saves time, as you can load

and unload one tray while the other one is in

the machine. A chiller plate with temperature

sensor allows the cooling of slides, e.g. to

keep spots moist.

NP2.1: Standard layout for 5 x 11 slides (up to 26

mm x 76 mm) and one sample plate

Slide tray removed from cooling chuck

System Platforms

MICROARRAYING

SUBSTANCES SUCCESSFUL-

LY DISPENSED BY GESIM’S

PIEZOELECTRIC PIPETTING

TECHNOLOGY:

Acetic anhydride, acetone

(propanone), acetonitrile

(ethanenitrile), betaine

(N,N,N-trimethylglycine), up to

3 M (up to 1.5 M for Pico-Tip),

chloroform (trichloromethane),

cyclohexanone, dextran solutions,

detergents (e.g. 0.02 % Genapol

C050, 2 % Triton X-100, 2% Tween-20),

dichloroben- zene, dichloromethane + 2 %

trichloroacetic acid, N,N-dimethylformamide (DMF), all mix-

ing ratios with water, dimethyl sulfoxide (DMSO), 1,4,-dioxane, DNA (PCR product < 2 kbp < 3 mg/ml in

buffer, plasmid < 4 kbp < 1 mg/ml), ethanol, ethylene glycol, moderate concentrations, N-FMOC-Ile-OH,

200 mM in DMF, up to 40 % glycerol in water, isopropanol (2-propanol), iodine in tetrahydrofuran/

pyridine/H2O 3:75:20:75 (v/v), liquid crystals (Merck Licristal® ZLI-2222-100 and Merck Licristal® MLC-

6681), MALDI matrix (alpha-cyano-4-hydroxy-cinnamic acid in NMP or 40 mg/ml 3-hydroxy-picolinic

acid / 6 mg/ml diammonium citrate in 20 % acetonitrile), methanol, 1-methylimidazole (N-methylimi-

Decks with vacuum or spring fixation exist,

but are only useful for pin spotting, as the

smooth touch-less operation does not move

slides. Decks holding microtitre plates (6 for

NP2.1, 12 for NP2.1/E) are available to pro-

duce microarrays in microtitre wells.

The standard slide tray has locating pins for

accurate slide positioning. Turning the bars

upside down creates a flat surface onto which

any substrate can be placed. And the space

between the bars can be magnetic (option),

helping to fix membranes by metal strips.

Of course, cooling of source plates and humid-

ification is possible to reduce evaporation and

to allow pipetting at the dew point..

Wash cycle in the normal (top) and the optional extra

wash station (bottom)

GeSiM’s piezoelectric tips are manufactured

from silicon and glass by micromachining

(reactive ion etching, anodic bonding etc.).

Samples come only in contact with silicon

dioxide (the Si surface is oxidized).

Each tip is individually actuated; sample

uptake and washing are done by syringe

pumps. The minimum dead volume (additional

sample) is approx. 1 µl. Unused sample can be

returned to the well after spotting. Various ar-

rangements on the pipette head are possible.

Tips are thoroughly rinsed on the inside and

the outside to avoid contamination. DNA solu-

tions (e.g. 0.1 mg/ml oligos) are washed away

in seconds. Proteins and peptides may need

longer, and in special cases additional wash

fluid (detergent, acid, base etc.) is taken from

a reservoir, a well or a second wash bowl.

Solids dissolved in organic solvents can be

spotted by putting a solvent “plug” and an air

gap between sample and system water.

Piezo tips for different droplet volumes

Nano-Tip J 0.35…0.6 nl

Nano-Tip A-J 0.2…0.4 nl

Pico-Tip J 0.05…0.08 nl

Special pipette tips e.g. Nano-Tip HV-J (for

highly viscous samples)

Nano-Tip H-J (heatable)

Nano-Tip AR-J (thinned for

printing into 96-well MTPs)

Two-row pipette head for up to 16 piezo dispensers

Piezoelectric Pipetting

GeSiM Piezo Pipettes

Slide deck, coolable micro-plate holders and

smoothly operating ultrasonic humidifier

www.gesim.de

GESIM Nano-PlotterTM

Stroboscope check of the piezo tips

Each piezoelectric tip is tested by analysing

the stroboscope image of the flying droplets

before and after spotting. If no droplet is

detected, maybe caused by an empty well,

the pipette is switched off and the array is

repaired later. (If dry spots are visible, a

microscope can search for missing spots!)

Droplet volume and speed depend on the

sample (mainly viscosity). Computing a body

of rotation from the droplet contour gives a

rough volume estimation; the speed is meas-

ured by analysing the droplet pattern at two

time points. You can thus optimize pipette

parameters so that all samples are spotted

with the same speed or the same volume.

If GesiM’s microfluidic flow sensor is placed in

the tube system, it can measure the flow dur-

ing the stroboscope test. This provides very

accurate droplet volume determination.

VIDEO

Probenaufnahme Dosierparameter Spotting Entleeren/Waschen Waschen mit Sample

aspiration

Optimising dispense

parameters and

functionality test

Additional rinse cycle

with extra wash fluid

(optional)

Spotting Emptying and

washing

Stroboscope image (left), 8-fold flow sensor on a

syringe pump unit (centre) and flow sensor graph

(right). A flow rate of 1.7 µl/min at a frequency of

100 Hz corresponds to a droplet volume of 280 pl.

Viscous samples and large volumes

GeSiM manufactures a variety of other dis-

pense heads. Viscous media can be delivered

by our heatable piezo dispensers. We also of-

fer heatable systems for bulk piezo pipetting

with large sample reservoirs. And finally there

is a solenoid valve dispenser for nanolitre

volumes.

Third-party dispensers with piezo or pneu-

matic valves can also be used; they work at

viscosities of up to a few thousand mPa⋅s.

And yes, even pins.

The Nano-PlotterTM syringe unit can also be

used for bulk displacement dispensing. Pas-

sive capillary tips or adapters for disposable

tips are available on request. Please consult

our Nano-Plotter catalogue for details and

more spare parts..

Special dispensing systems from GeSiM. Left, heatable piezo tip that is compatible with microtitre plates, with

additional temperature sensor cable in a Nano-Plotter. Centre, heatable bulk piezo dispenser with reservoir

(without fluidics). It can deliver droplets as small as 10 pl. Right, glass capillary dispenser with solenoid valve for

higher viscosity and volumes of > 50 nl. Needs a fluidic system with slight overpressure.

Pipetting Cycle

MICROARRAYING

Special Equipment and Software

Software and programming

The intuitive Nano-PlotterTM control software,

NPC16, supports all configurations, but you

don’t have to care for this, as the system

comes pre-configured.

NPC16 features a scripting tool (Nano-Plot-

ter Language) with arithmetic expressions,

constructs like loops and branches, and full

hardware access; so you can develop your

own “NPL programmes”. This is an option, but

rarely needed, as all necessary programmes

are included, ready for use.

Standard procedures

Service routines exist for overnight standby

with occasional washing to keep pipettes

happy, a cleaning procedure for dirty pipettes,

or semi-automatic adjustment of pipettes to

the same droplet speed, to name a few.

Our standard spotting routines should address

all your needs no matter how pipette head or

targets are set up, require zero programming

and feature extensive data logging for quality

control, including saving of stroboscope

pictures and determining droplet volumes and

speeds before and after spotting.

Transfer list: Programmes are not restricted to

a certain order (as in the left two panels). In

fact, sample wells can be assigned to arbitrary

spot positions in one or more sub-arrays, also

with replicates. This is described in a “transfer

list” text file, but you can use a graphical user

interface by clicking on source and target

positions. But given the many options, typing

is often easier than clicking.

Sequential spotting: ”Drop on demand” allows

to spot only one sample at a time. Although

slow, this flexible mode allows any array

layout and adjustment of inter-tip positions.

Spotting with automatic target finding via a

microscope must always be done sequentially.

Simultaneous spotting: This is the fastest

method, as all tips fire at the same time. Spots

must be arranged in the pitch of the pipette

tips (9 or 4.5 mm), so spots in between are

printed in subsequent cycles. Array positions

are again given by a (different) transfer list.

Thanks to our customers, our “TransferSim”

programme has matured to a “Swiss Army

Knife” with countless options, e.g. elaborate

wash procedures, sample uptake using only

a subset of pipettes, spotting only on certain

slides or sub-arrays, solvent delivery, ”spot-

ting on the fly”, varying pipetting parameters

and much more. Multiple source plates, with

or without manual exchange, are supported.

Parallel spotting is also the only choice when

spotting into MTP wells.

Spotting onto tiny structures

It is not easy to dispense a 60 µm droplet

exactly onto a target of similar size. A micro-

scope camera on the print head helps: first

spot positions on all targets are identified

(typically via alignment marks), then the X/Y

position of the droplet is measured via a test

spot to correct even the smallest deflection.

The pattern recognition works well for pure

two-dimensional targets such as microe-

lectrode pads or straight conducting paths,

but also for nano-wells, microcantilevers,

microfluidic channels and more. Missing spot

detection and repair is also possible; live spot

detection during printing on a light table will

soon be available.

This software extension allows the unlimited definition

of spot positions relative to alignment marks. On

the right: microscope above a chip and example of a

successfully detected microelectrode array.

Handling of thousands of samples

All Nano-Plotters can be combined with

plate hotels. This here from Thermo allows

automatic handling of up to 56 MTPs. Plate

storage is temperature- and humidity-con-

trolled.

A lid handler guar-

antees the transfer

of covered MTPs,

especially for quickly

evaporating samples.

www.gesim.de

GESIM Nano-PlotterTM

Examples from Research and Manufacturing

0,1 mg/ml Oligo

10 drops = 3 nl

100 drops = 30 nl

2 m

m

Arrays for DNA, proteins and more

GeSiM’s piezo technology provides high spot

homogeneity and allows different spot sizes

simply by varying the number of drops per

spot. Different samples can be spotted by ad-

justing pipetting parameters (mainly voltage).

For higher viscosity, “high-viscosity” tips

(“HV-J”) and heatable tips are available.

The spotting pitch depends on the surface.

Hydrophobic substrates allow grid sizes down

to 150 µm. Piezoelectric tips are perfect to

process typical buffers such as 3x SSC, phos-

phate-buffered saline (PBS) or Tris, but also

up to 40% glycerol.

The Z-height sensor measures all target Z-positions

for automatic adjustment of spotting heights

Spotting onto microstructured substrates is

possible. Using piezoelectric, solenoid valve

or displacement (“passive”) dispensing, nan-

olitre and microlitre volumes can be spotted

onto e.g. conducting MALDI targets. Automatic

centring is possible, if image processing is

used (see above).

Microarrays can be generated in three-dimen-

sional objects, e.g. MTP well bottoms.

Spots on a membrane (2.5 nl, 0.4 mm pitch) and in a

96-well MTP at 0.3 mm pitch

Large-scale chip production

Of course, the rather small Nano-PlotterTM

is ideal for research and chip development,

but this does not mean that it is incapable of

high-volume chip production.

The picture on the right shows a former

production site for specially designed FDA-

cleared diagnostic chips of a company in Utah.

And a growing number of companies in China

rely on the Nano-PlotterTM for bulk biochip

production.

Sensors

Numerous labs all over the world use the

Nano-PlotterTM for special operations such as

the production of (bio)sensors, where (bio)

molecules are spotted onto small electrodes,

optical structures, microcantilevers or similar.

This requires very precise positioning that is

aided by a microscope and automatic image

processing (here an example from Scotland).

The microscope can also be used in combina-

tion with other dispensing systems such as

pin tools or a dispenser for adhesives.

Gesellschaft

für Silizium-Mikrosysteme mbH

Bautzner Landstraße 45

01454 Radeberg, Germany

Tel. +49–351–2695 322

Fax +49–351–2695 320

info@gesim.de

For more information (applications,

systems, distributors etc.) please visit

www.gesim.de

Specifications subject to

change without notice

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GESIM Nano-PlotterTM

We are not licensed under any patents owned

by Oxford Gene Technology Limited (OGT) or

related companies and cannot pass any such

licence to our customers. A licence under

OGT’s patents may be necessary to manufac-

ture or use oligonucleotides arrays.

GeSiM mbH