Category Archives: Non-Contact-Microarrayer

Nano-Plotter

Platforms / Sizes

The modular non-contact Microarrayer

The Nano-PlotterTM NP2.1 is available in two sizes:

NP2.1 with a slide capacity of 55; NP2.1/E with a slide capacity of 120.

All Nano-Plotter versions come with the following common features:

  • 1 to 16 independent piezoelectric pipetting tips in any combination. Post sales upgrade is possible.
  • Sample aspiration from 96 well or 384 well micro plates
  • Array density > 3000/cm2
  • Dust cover
  • Automatic wash/dry station
  • Optical function test of each individual pipette tips after sample aspiration

 

 

Easy and superb arraying onto slides (For HD introduction video click here)

 

NP2.1 can be configured to a particular application. And… it can grow! It is easy to increase the number of pipetting channels after sales. Also, most accessories can be upgraded. Thus, this instrument might be your companion from research to production.

 

Pipetting Tips

Non-contact Micro Dispensers work on any Substrate Surface

The Nano-PlotterTM piezoelectric pipetting tips for the Picolitre range consist of glass and silicon- they are valveless and fabricated from microstructured glass/silicon wafers. Alternatively the instrument is available with solenoid valve dispensers for the Nanolitre range.

The tips interface with the instrument’s fluidic system through metal shafts. Each pipet connects to a dedicated syringe that supplies both sample and washing fluids. All N2.x instruments can be equipped with 1-16 tips in any combination.

Due to the nature of non-contact dispensing the Nano-Plotter supports so called Spot-On-the-Fly mode (SOF, since 2017) for microarraying applications with only one drop per spot position. Particularly when processing a larger number of flat substrates (E.g. glass slides) SOF saves up to 50% time.

Piezoelectric Tips for the Picolitre range

  • Nano-Tip J:     0.35…0.4 Nanoliter
  • Nano-Tip A-J: 0.25          Nanoliter
  • Pico-Tip J:       0.06          Nanoliter
  • Nano-Tip J-H: 0.35…0.4 Nanoliter (External heater unit required)
  • Pico-Tip J-H:   0.06          Nanoliter (External heater unit required)

Tips with special design:

  • Nano-Tip HV-J: For high-viscous samples
  • Nano-Tip AR-J: Slim design,for array printing into 96-well plates

 

 

Fast solenoid valve on the Nano-Plotter (SOF mode)

 

Solenoid Valve Pipetting Tips (Option):

  • 40…350 Nanolitre drop volume
  • Affordable glass nozzle for low operational costs
  • Upgrades available for all Nano-Plotter types

 

 

Liquid Management for Piezoelectric Tips

 

The Nano-Plotter piezoelectric pipetting tips consist of glass and silicon.

Process Control

Automatic Function Test for each Piezo Tip

A camera system with sophisticated image analysis inspects each piezoelectric pipettor after sample aspiration. The analysis is for:

  • Deflection of the main droplet from the vertical
  • Droplet size, count and speed
  • Deviation of satellite drops from the main jet

The checking function returns a result (Path/Fail) for each pipette. So if a tip fails to spot according to specification, the Nano-Plotter can either repeat sample aspiration, or continue printing with only the working pipettes and repair the missing spots afterwards.

 

strobocheck-camerastrobo_goodstrobo_bad
Stroboscope cameraValid droplet image. The jet angle is inside a user defined region, the number of satellite droplets is small enough.Invalid droplet image. The flight angle of one satellite droplet differs too much from the angle of the main jet.

A more accurate drop volume estimation is available with the optional micro flow sensor.

Target Trays

Customized Trays are available on Request

For fast and easy replenishment of targets, standard Nano-Plotter trays adopt both 1" by 3" slides and membranes. Four strips with locator pins separate the slides from each other and inversion of the strips gives a smooth area for membrane arrangement.

Customized shapes are available on request.
SlideTray

Volume Measurement

Unique Drop Volume for different Sample Species

NP21_Flow-Sensor

NP21_Flow-Sensor

Naturally, the dispense behaviour of the piezoelectric tips is affected by the sample. A highly sensitive flow sensor in the system liquid path of each tip measures the compensated volume of the ejected drops.

Chilling/ Humidifying

Management of Rare Sample Species

External units for chilling and humidifying allow establishment of conditions close to the dew point. The ultrasonic humidifier connects directly to the hood enclosure of the instrument, thus even microliters of samples can remain on the deck of the instrument with minimum evaporation losses.

Coolable_tray_microwell_plate

Both the coolable base plate underneath the slide tray and the microplate holder can be connected to a recirculating chiller to control the temperatures of both the samples and the targets.

 

TwinTip – Pipetting and Mixing

TwinTip adapter for the Nano-Plotter

TwinTip adapter with two solenoid valve micro pipets

TwinTip adapter with two solenoid valve micro pipets

 

Chemical reactions at a microscale and hardening of two-component systems are just two applications for the new TwinTip adapter for the Nano-Plotter. In contradiction to other micro mixers the TwinTip adapter allows to aspirate tiny volumes of two different species from a standard micro well plate.

 

 

 

 

 

 

 

 

 

 

 

 

The TwinTip adapter accomodates the native GeSiM piezoelectric pipets as well as solenoid dispense valve pipets. Following setups are possible:

  • Two GeSiM piezoelectric pipets with drop volumes between 60 pL and 400 pL
  • Two solenoid valve pipets with droplet volume of 50 nL
  • One GeSiM pipet, one solenoid valve

The TwinTip adapter toggles the pipets between two positions: OPEN (Aspiration/ wash position) and CLOSE (Dispense position). Simultaneous drop release at the CLOSE position leads to a perfect mix up of both drops on the target surface due to the high kinetic energy of the drops.

 

Handling of the TwinTip adapter

Handling of the TwinTip adapter

Stroboscope view of collision of microdrops

Stroboscope view of collision of microdrops

More than a Microarrayer

Customized Versions of the Nano-PlotterTM

Besides array spotting, GeSiM has the flexibility and capability to create custom instruments on request. The Nano-PlotterTM software comes with an open user interface for the development of liquid handling applications by the customer.

Third party dispensers and other tools on the Nano-Plotter print head

Third party dispensers and other tools on the Nano-Plotter print head

In addition, GeSiM develops and delivers OEM Versions of the Nano-PlotterTM to other instrumentation manufacturers.

- Heatable piezodispensers
- Dispensers with reservoir cartridges on top
- Dispensers for high-viscosity media
- Passive Microliter dispensers
- Plate hotel for large samples sets
- Vacuum tweezers
- Wash system for dedicated cleaning solutions
- Print head camera for process observation

As an example, microarray spotting can be accomplished by Microliter liquid handling or manipulation of mechanical parts like glass lids. For manufacturing of a large number of identical arrays with a limited set of samples we recommend piezoelectric tips with an integrated reservoir.

Benchtop lab automation exceeding liquid handling is available with the GeSiM BioSynthesizer.

 

Benefits

Optical Target Recognition

The Optical Target Recognition (Option) comprises a camera system assembled to the print head as well as a software extension for the Nano-Plotter software.

Considering a typical drop diameter of 50 Microns the Nano-Plotter is well appropriated to apply Nanolitre amounts of liquid to target structures in the same range. However, the nozzle alignment above tiny targets may become challenging if attempted manually.

Microfluidic Chip with tiny dispense targets

Patterned chip layout

Alignment Camera for automatic nozzle adjustment

Alignment camera

The Optical Target Recognition automatically detects small patterns such as microelectrodes, nanowells and microcantilevers. This enables the Nano-Plotter to precisely find positions and spot onto them (manual selection of points by clicking in the camera window is also possible).

The camera looks for two alignment marks (“Fiducials”) on each chip/substrate. Spotting positions can be defined relative to the alignment marks. Regular target patterns like arrays of small wells usually don’t require dedicated fiducials.


Click here for a customer example…!

 

Procedure

Basic Function of Target Camera

The fiducial pattern recognition camera allows both point-and-click definition of the spot layout in a live video image or automatic target recognition by image processing software.

 

The video shows how the alignment process works. The user needs to write a “spotting plan” in advance for each spot pooint.

1. Initially a precise offset measurement gives the exact distance between the camera axis and the drop trajectory of each dispenser.
2. Now the camera travels to each of the two alignment markers on each target substrate. Two pictures are taken of each target substrate.
3. The software calculates the coordinates for each spot point and completes the spotting run without any further user interaction.

Microarrays and Service

Customer Service is a Priority with us!

Fluorescence Image of a Microarray

Fluorescence Image of a Microarray

Microarraying applications are rather complex. The result depends on a lot of laboratory steps and many SOPs while all the necessary instruments are expensive.

Regarding the spotting part of each microarray experiment we will work with you towards your application goals. Don’t place an order on such instrument before you are sure it does the job. If available we can accept your printing material for free feasibility tests. Visits to our premises as well as phone/Skype conferences can also transfer our experience to you.

To a certain extent GeSiM also offers microarray spotting services providing non-commercially available reagents/samples/substances can be supplied to us.

We are often asked about throughput for production projects: It will very much depend on your array layout and other parameters. Please contact us with your particular idea.

 

Manufacturing of Array based Diagnostics

Multiplexing your Assay

Until now diagnostic kits for blood or patient serum are mostly based on nitrocellulose membrane. The analyte hybridizes against a limited set of test molecules and calibration standards, typically less than five.

By switching to the microarray format much more parameters can be tested. In recent years the Nano-PlotterTM NP2.1 has been proofen as reliable tool for the production of microarrays for diagnostic use.

Please download this pdf for selected reference installations.

 

Arraying into 96-well plates

Multi Parameter Analysis with 96 Well Plates

Nano-Plotter NP2.1 instruments can be configured for arraying on the bottom of 96-well microplates. On request special target decks and 2-row print heads are available, matching the 9 mm pitch. GeSiM has developed an optimized piezoelectric pipet (Nano-Tip AR-J) with slimmer design for this application. The highest achievable spot density is significantly dependent on sample type and surface properties of the plate.

Array_in_96wellplate

64 spots of different size made with Nano-Tip AR-J368 spots
1 drop (250 pL)5 drops (1.25 nL)10 drops (2.5 nL)1 drop

This example was printed with fluorescine solution containing 10% glycerol.We used a 96 well plate from GreinerBioOne (Order #762070, Midbind ).

Slidedeck_for_MTPs

NP2.1/E tray for 12 standard format well plate

 

Antibody Arrays without Cross Contamination

Antibody Pipetting with Piezoelectric GeSiM Tips

  • 4 antibodies were assessed (IL6, EGF, PSA, C-RP)

    Rows of protein spots before and after tip washing

    Rows of protein spots before and after tip washing

  • Antibodies were spotted at 100ug/mL concentration
  • To evaluate carry over between each antibody two plain water samples were aspirated (Water Wash 1 and 2)
  • The GeSiM Nano-Tip was used at one drop (approx 350 pL) per spot
  • Arrays were spotted on GenTel PATHTM slides
  • The tip was washed with water (9 seconds), 0.2N KOH (2 seconds) then water (9 seconds). The piezo was activated during the  water washes
Bar graph quantifying signal intensities

Bar graph quantifying signal intensities

Bar graph quantifying percent CVs

Bar graph quantifying percent CVs


Courtesy of HTS Ressources, San Diego (USA)


 

Common Recommendations for Spotting of Antibodies:

  • Use protein concentrations of max. 1 mg/mL containing less than 1 M salt.
  • As proteins need higher piezo voltage, activate the stroboscope break in the standard NPL programs to adjust the parameters. In case of varying spotting parameters for different samples optimized values should be added to the well plate file (See manual for more instructions).
  • Carbohydrates like trehalose can help hydrate proteins and maintain their native structure even in a dry state. But you must prove that these viscous solutions can be spotted without problems.
  • If you have a large protein supply, ultrafiltration would remove aggregates. If you have only small volumes, centrifuge at least.
  • Avoid to suck particles into the pipettes by not using up the entire sample volume.
  • Added buffer should be sterile-filtered to prevent spoiling.
  • The first spot in a row of spots may be stronger than the following ones. If this is the case, define a “yellow paper object” near your first slide and dispense the first spots onto this target.
  • Adjust the piezo parameters for each pipette before each run until the droplet pattern in the stroboscope “looks good” for all of them. Your experience is needed here.
  • If you require low inter-tip CVs or need to know absolute droplet volumes, dispense labeled protein and quantitate spots in a scanner.

Cell Lysate Microarrays (Reverse Microarrays) – From Cells to Protein Profiles

Reverse Phase Protein Arrays

Microarrays with individual cell lysate spots of a 130 µm diameter, representing 1 to 10 cell equivalents each, are being produced using the robust piezo-electric non-contact deposition method of the Nano-Plotter. Signals are generated by fluorescently labeled antispecies antibodies. Due to the extremely low sample consumption of the spotting process large numbers of replicate arrays can be produced and thus allow the efficient determination of substantial numbers of proteins.  The simple, robust and highly parallel architecture of the assay yields precise quantitative information and allow time course monitoring of protein expression and activation.

From tissue to data (Source: Zeptosens AG)

From tissue to data (Source: Zeptosens AG)

 

From tissue to data: The procedure of Zeptosens Cell Lysate Microarrays

 

 

 

 

 

 

The applicability of the ZeptoMARK CeLyA approach has been demonstrated among others by

  • Identification of disease relavent marker proteins in cultured cells, tissues, microdissected material as well as depleted serum and urine
  • Determination of dose efficacy and IC50 of drug candidates
  • Quantification of cell signalling pathway activation / inhibition with a precision of better than 20%
  • Monitoring phosphorylation changes on multiple kinases

More information on this technology ist available from Bayer Technology Services, Leverkusen (German)


 

Glycoprotein Arrays

Protein Microarrays

to study Post Translational Modification Changes as a Function of Disease (Tasneem et.al.)

Background

Complementary to the popular antibody arrays, this application uses pre-separated proteins from cellular lysates or other bio-fluids. The resulting arrays are probed with agents that can specifically detect certain post translational modifications. Differences in modifications between different disease states can therefore be highlighted.

Method

Glycoproteins from serum samples can be enriched and separated by nonporous reversed-phase HPLC. Separated proteins are then arrayed on nitrocellulose slides and probed with multiple types of lectins using a biotin-streptavidin platform to detect glycan structures present in the glycoprotein.

Dilution series of standard glycoproteins to study lectin specificity.

Dilution series of standard glycoproteins to study lectin specificity.

 

Printed glycoproteins were probed with biotinylated lectin followed by streptavidin conjugated to green florescent Alexafluor.

 

Comparison of different glycan structures in sera from healthy patients and those diagnosed with pancreatic cancer using multi-lectin detection.

Comparison of different glycan structures in sera from healthy patients and those diagnosed with pancreatic cancer using multi-lectin detection.

Each vertical panel of spots represents a unique peak from the reversed-phase HPLC separation. Each separated glycoprotein was printed in 9 replicates to monitor variations due to printing. Within 10% variation was found for most fractions studied.

 

 

 

 

 

 


Courtesy of: Tasneem Patwa
University of Michigan Medical Center
Department of Surgery
MSRB I, A510B
1150 W. Medical Center Drive
Ann Arbor, MI 48109-0650
USA


Patwa TH, Zhao J, Anderson MA, Simeone DM, Lubman DM.: Screening of glycosylation patterns in serum using natural glycoprotein microarrays and multi-lectin fluorescence detection. Anal Chem. 2006 Sep 15;78(18):6411-21.

 

 

Stent Coating

Coating of Drug Eluting Stents

The GeSiM Nano-PlotterTM has been extended by a motor controlled rotor for the application of Micrograms of solved polymer on cylindrical microstructures. This application notice shows how to dispense Nanoliter amounts on stain less steel made coronar stents. Basically a “stent” is a tiny mesh or scaffold for insertion into coronar vessels of the human body. Orginally stents were developed to prevent blood vessels from collapsing after ballon angioplasty. A resistent problem was that bar metal stents still experienced reblocking as a body’s response to the “controlled injury” of arterial vessels during insertion of the stent. The controlled drug release requires to embed the drug into a soluble polymer. For a reproducible release rate an exact amount of drug solution has to be applied to the scaffold structure of the stent. Dipping the stent into a reservoir is no appropriated approach. The rather high viscous drug solution simply bridges meshes of the stent structure which leads to an unpredictable amount of remaining drug after levering the stent out of the reservoir. Placing the stent onboard of the micropipetting system Nano-Plotter brings up several advantages:

  • Less waste of material by controlled aspiration of sample into the dispenser nozzle
  • Applying of single drops below one Nanoliter avoids bridges between and tears on the stent structure
  • The programmable number of microdrops ensures exact and reproducible drug deposition solely on the stent structure
  • The camera based alignment of the dispenser above the stent ensures that no stent will be missed
  • The rotating stent holder allows homogenous coating of all sides of the cylindrical stent
The rotating stent holder can be moved to any position between 0° and 360°

The rotating stent holder can be moved to any position between 0° and 360°

A single drop volume is about 250 Pikolitres

A single drop volume is about 250 Pikolitres

 

The GeSiM Nano-PlotterTM has been extended by a motor controlled rotor for the application of Micrograms of the operation software of the Nano-Plotter allows two different modes for dispensing on the stent structure: a) Continuous line dispensing emitting drops at a fixed frequency (faster) b) Step-by-step dispensing, the nozzle stops before ejecting a drop (more accurate).


The development of the rotating stent holder was initiated by

UoG-logo.gifUniversity of Greenwich, School of Sciences, Medway Campus, Central Avenue
Chatham Maritime, Kent, ME4 4TB, UK
We thank Dr. Dionysios Douroumis and his group

 

Loading of Biosensors

Electrical Biochips – The comfortable Way of detecting Biomolecules

Eight channel head over biochip wafer

Eight channel head over biochip wafer

 

The department “Biotechnical Microsystems (BTMS)” of the Fraunhofer ISIT is one of the worldwide leading groups in the field of electrical biochip technology. The electrical biochips offer intrinsic advantages because of particle tolerance and mechanical robustness by the direct transduction of biochemical reactions into electrical current.

The use of gold electrode arrays combined with integrated reference and auxiliary electrodes along with sensitive, selective measurement techniques like “Redox-Cycling” enables powerful sensor systems. These arrays are useful for the detection of a variety of analytes within one sample simultaneously. User-friendly operability is realized by integrating the biochips into cartridges. In combination with micro-fluidic components and integrated electronics, these electrical microarrays represent the basis of rapid and cost-effective analysis systems. They can be used to identify and quantify DNA, RNA, proteins, whole cells as well as haptens.

 

Droplets on a biochip

Droplets on a biochip

The used array chips consist of 16 gold electrodes for immobilizing capture molecules and the final electrochemical read out. These positions are 350 µm in diameter and therefore the capture molecules have to be deposited position specific by a piezodriven nanodispensing device. In our case we use the nanospotter technology from GeSiM (Left image). The chips are spotted directly on a diced wafer with 316 biochips glued on a dicing frame. Each chip will be picture recognized by the nanoplotters head camera before dispensing. Then the chips are spotted individually by 4 or 8 nanotips with a dispensed amount of 10 nl on each chip position. Afterwards the head camera takes a picture of each freshly dispensed chip for quality control (Right image). Then the chips are incubated, blocked, dried and are ready for assembling.


FhG-ISIT

FhG-ISIT

 

With Courtesy of Dr. Eric Nebling, Fraunhofer Gesellschaft Germany (ISIT)
Fraunhoferstrasse 1, D-25524 Itzehohe

 


–     L. Blohm et. al.: “Rapid detection of different human anti-HCV immunoglobulins on electrical biochips”, Antibody Technology Journal, 2014:4 23–32

–      S. Kraus et. al.: “Quantitative measurement of human anti-HCV Core immunoglobulins on an electrical biochip platform”, Biosensors and Bioelectronics 26 (2011) 1895 – 1901