Category Archives: Nanoplotter-Applications

Nano-Plotter

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