IMAPlateTM 5RC96 - world's first miniaturized analytical platform

IMAPlateTM 5RC96 a Product from

Patented Intelligent Multifunctional Analytical Technology

April 2012

© 2012 NCL New Concept Lab GmbH

Content

• Introduction – Working principle

– Classical liquid handling

– Technical data

• Application – Liquid tansfer

– Measurement

– In-well homogeneous assay

– In-well heterogeneous assay

– 3D cell culture

• Summary

© 2012 NCL New Concept Lab GmbH 2

Introduction of IMAPlateTM 5RC96 http://www.youtube.com/IMAPlateTM

A standard 96-well plate formatted

disposable miniature multi-utility lab device

with Integrated functions of

Pipette, Test Tube, Cuvette ...

The world's first analytical platform

capable of manually performing parallel, high throughput

liquid transfer, reaction, analysis and assay.


http://www.youtube.com/IMAPlateTM

IMAPlateTM 5RC96 Working Principle – Capillarity


illustrations of unique self-dosed capillarity liquid handling (single unit)

capillarity chamber

non-capillarity reservoir The capillarity chamber is able to use capillary action to suck

up quantitative amount of solution from the bottom opening and to keep the dosed solution in the capillarity chamber.

The solution can be collected into a 96-well plate by centrifugation or absorbed by using a filter paper . The solution can be measured

in a 96-well plate reader.

4

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Illustrations of Pipetting Solution to IMAPlateTM 5RC96

methods for pipetting 1- 5µl or <1µl of solution


methods for pipetting 15-25µl of solution

adding ragents and mixing pipetting reaction solution sealing the bottom open adding reagent B mixing and in 96-well plate to IMAPlateTM 5RC96 and adding reagent A removing the seal

assay measurement in-well assay and measurement

manual pipetting manual multi-pipetting automated workstation <1µl of solution pipetting

5

IMAPlateTM 5RC96 Technical Data

comparison between IMAPlateTM 5RC96 and Nunc. MaxiSorp F96 plate


IMAPlateTM 5RC96 Nunc. MaxiSorp

Plate Format SBS 96-well format SBS 96-well format

Well Dimension (Φ x H) 1.1mm x 5mm 6.9mm x 10.7mm

Total volume 5μl (or 50μl) 360μl

Working volume (V) 5μl (or 15 – 25μl) 100μl

Surface area of working volume (Sa) 18mm2 (60 + 34)mm2

Surface area to volume ratio (Sa/V) 3.6mm2/μl 0.94mm2/μl

Initial binding velocity d[AB]/dt (B Sa/V) 3.8 kon[A][B] 1 kon[A][B]

Complex density [AB] (B Sa/V) 3.8 Keq [A][B] 1 Keq [A][B]

Diffusion distance from center to wall (X) 0.55mm 3.5mm

Diffusion time (X2 = Ddif•t) 0.3/Ddif 12/Ddif

Light path length 5 or 7.5mm (5 or 25μl) 3mm (100μl)

6

Applications of IMAPlateTM 5RC96

96 channel pipette & replicator for simultaneous liquid transfer sampling, dilution, spotting, inoculation, …

96 micro-cuvette for UV-Vis-IR full spectrum measurement with easy

sample recovery option DNA, RNA and protein quantification at UV, compound analysis at UV-Vis-IR,

assay result readout to enhance sensitivity.

96 micro-well plate for reactions and assays

In-well homogeneous assays (e.g. miniature, high sensitivity assays) In-well heterogeneous assays (e.g. miniature ELISA) Solid phase enzymatic or binding assays for compound, ligand screen 3D cell culture and cell based assays Molecular biological assays Proteomics applications Multi-step solid phase chemical synthesis










Illustrations of sampling, dilution, spotting, inoculation…


touch-load solution

prepare a master plate

spin to new plates for sampling, dilution and ...

spot on membrane for dot blot assay and ...

add to cell cultures in 96-well plate for making replication, screening compound and ...

9









Illustrations of assay in 96-well plate and measurement in IMAPlateTM 5RC96


perform assay in 96-well palte transfer assay solution to IMAPlate measure IMAPlate in a reader

11

The use of two wavelength absorbance measurement to calculate the absorbance for IMAPlateTM 5RC96

IMAPlate measurement

Due to small diameter of the capillarity chamber, the observed Abs is the sum of absorbance of the solution and blockage of incident light, which causes the baseline shifting upward. Assuming the blockage is the same over the spectrum, the absorbance can then be calculate by following equation (2).

Absorbance = AbsPeak – AbsBaseline (2)


96-well plate measurement

Absorbance = - logT = - log(I/I0) (1) The absorbance of the solution in 96-well palate is calculated according to the equation (1), where I0 is the intensity of incident light and I is the intensity of transmitted light.

I0 I

I0 I

0

0.5

1

1.5

2

2.5

3

350 400 450 500 A

bsorb

ance

0

0.5

1

1.5

2

2.5

3

350 400 450 500

96-well plate wavelength (nm) IMAPlate wavelength (nm)

Abs

Abs

Abspeak

Absbaseline

Absorb

ance

Abspeak

12

High sensitivity for absorbance and fluorescence measurement resulted from long light path-length of IMAPlateTM 5RC96


Absorbance of 5µl dye solution in IMAPlateTM 5RC96 = 60µl in 384-well plate = 175µl in 96-well plate

Fluorescence count of 5µl fluorescein solution in IMAPlateTM 5RC96 = 85µl in 96-well plate

0

0.1

0.2

0.3

0.4

0.5

0.6

0 50 100 150 200 250

IMAPlate 1-5µl

IMAPlate 15-35µl

384-well 25-100µl

96-well 50-225µl

Absorbance vs Volume

(comparison of IMAPlate, 384- and 96-well plates)

A

bs450-A

bs650

volume of dye solution (μl)

Fluorescence vs Volume

(comparison of IMAPlate and 96-well plates)

Flu

ore

scence c

ounts

volume of fluorescein solution (μl)

* Data obtained from PerkinElmer EnSpire® Multimode Plate Reader.

0

20000

40000

60000

80000

100000

120000

140000

0 25 50 75 100 125 150

IMAPlate bottom reading 2.5&5μl

96-well plate top reading

13

High-throughput small sample volume quantification by touch-loading from a 96-well plate

DNA and protein measurement at UV


DNA (µg/ml) Abs SD CV%

0 0.000 0.001

3.6 0.028 0.003 10.9

7.3 0.071 0.003 4.3

14.5 0.160 0.001 0.6

29 0.329 0.004 1.2

58 0.632 0.006 1.0

116 1.239 0.015 1.2

232 2.449 0.004 0.2

23 0.256 0.011 4.3

BSA (mg/ml) Abs SD CV%

0 0.000 0.001

0.16 0.045 0.001 1.6

0.31 0.105 0.004 3.4

0.63 0.194 0.006 2.9

1.25 0.382 0.017 4.4

2.5 0.767 0.041 5.4

5 1.429 0.079 5.5

10 2.462 0.003 0.1

2 0.641 0.008 1.3

0

0.5

1

1.5

2

2.5

0 50 100 150 200 250

dsDNA Std Curve

23µg/ml dsDNA sample

0

0.5

1

1.5

2

2.5

0 2 4 6 8 10 12

BSA Std Curve

2mg/ml BSA sample

Ab

s26

0-A

bs3

50

dsDNA concentration (μg/ml)

Ab

s28

0-A

bs3

50

BSA concentration (mg/ml)

14

Good correlation with other measuremnt method

• DNA measurement by pipetting to IMAPlate linear increase in Absorbance vs volume in the range of 1 to 5.5μl.

good correlation with nanodrop.


Absorbance vs Volume

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 1 2 3 4 5 6

volume (μl)

Ab

s26

0-A

bs3

50

y = 2.3306x R² = 0.9992

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0.0 0.5 1.0 1.5 2.0

Abs260– Abs350 (5μl in IMAPlate)

OD

(N

ano

dro

p)

Correlation (Nanodrop and IMAPlate)

15

To increase sensitivity of ELISA by reducing substrate volume and transferring the solution to IMAPlateTM 5RC96 for measurement

Abs =E l C

• According to Beer-Lambert Law, light absorbance is the

multiplication of absorption coefficient (E ), path length (l) and concentration (C).

• With same amound of enzyme in the well, the less volume of substrate solution, the higher concentration of colored product and the shorter path length for colored solution.

• Abs will not increase when using less substrate and measuring in the same well because the shorter path length cancels out the higher concentration of colored product.

• Once the colored solution is transferred to IMAPlate for measurement, the Abs increases due to the the higher concentration of colored product and the longer path-length of IMAPlate.


Human IL6 ELISA

(15μl TMB & 5μl stop solution with touch-loading)

Human IL6 concentration (pg/ml)

0 5 10 15

Abs

45

0-A

bs

65

00

0.5

1

1.5

2

2.5

3

U-IMAPlate

Nunc F plate

100 200 300 400 500 600

Sample

Spies P. et al. A simple approach to improve the sensitivity of enzyme-

linked immunosorbent assay: Using the IMAPlate 5RC96 for result

readout, Analytical Biochemistry, 397 (2010): 48-55.

E

l XC

E

l C

E

1/Xl XC

E l C E l C XE l C

16

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To increase sensitivity for commercial ELISA kit (pre-coated plate)

Procedure

• Prepare a set of extanded standard with additional several lower concentration points.

• Perform the ELISA according to the protocol in the kit till to the step before the addition of substrate solution. Then follow the procedure below.

1) Add 40μl of TMB solution instead of 100μl to the wells of ELISA kit plate.

2) Shake the plate on the plate shaker with 700-800 rpm for 15-20min.

3) Pipette 10μl of the stop sulotion to the wells to stop the reaction.

4) Transfer 40μl of the reaction mixture to the non-capillarity reservoir of the IMAPlate.

5) Tap the IMAPlate several times horezontally to facilliate solution go to the capillarity chamber.

6) Measure the IMAPlate at 450nm and 650nm in a plate reader.

7) Calculate the true Abs by substrating Abs450 by Abs650 for plotting.


Human IL-6 (pg/ml)

0 100 200 300 400 500

Abs4

50

- A

bs6

50

0

1

2

3

4

Kit plate

IMAPlate

Human IL-17A (pg/ml)

0 500 1000 1500 2000

Ab

s4

50

- A

bs6

50

0

0.3

0.6

0.9

1.2

1.5

1.8

Kit plate

IMAPlate

LEGEND MAX™ Human IL6 ELISA

LEGEND MAX™ Human IL17A ELISA

17









Illustrations of in-well homogeneous assay

5μl low volume assay with add-mix-meaure protocol


place IMAPlate upside down pipette 2nd assay solution inverte IMAPlate to mix and pipette 1st assay solution and measure in a reader

temporally seal the bottom open pipette 2nd assay solution, peel off the bottom seal, allow and pipette 1st assay solution mix and incubation solution to go down and measure

15-25μl macro volume assay with add-mix-measure protocol with incubation

19

In-well miniature protein quantification assay

5µl Bradford assay 25µl BCA assay 4µl of protein and 1µl of Bradford reagent 2.5µl of protein and 22.5µl BCA working reagent

mixed in the capillarity chamber by inverting incubated 30min at 37oC on a shaker in the non-

the IMAPlate and measured immediately. capillarity reservoir of a parafilm sealed IMAPlate.

Peeled off the parafilm and measured.


0.0

0.1

0.2

0.3

0.4

0.5

0.6

0 20 40 60 80 100 120

0.0

0.5

1.0

1.5

2.0

2.5

0 0.5 1 1.5 2 2.5

Ab

s59

5-A

bs8

00

BSA concentration (μg/ml)

Ab

s56

2-A

bs8

00

BSA concentration (mg/ml)

20

CALB concentration (ug/ml)

0 10 20 30 40 50 60

Abs

405

- Abs

500

0

0.5

1

1.5

2

Nunc plate 200ul

IMAPlate 20ul

In-well miniature enzyme assay (15-25µl)

• e.g. Candida Antarctica Lipase B assay in IMAPlate for rapid colony screening *M. A. Sciotti et. al. IMAPlate Based Miniature, High Sensitive, Rapid Screening Method for Detecting Bioengineered, Secreted Lipase Activities in

Yeast Expression Systems. CHIMIA, Volume 64, No. 11, November 2010, Pages 789-792


pick up individual inoculate to 96-well plate test enzyme activity in plot Std curve whole clony for protein expression medium and lysate for data analysis

only 1/10 assay volume of 96-well plate

higher sensitivity

reducing assay cost

minimizing upstream processing scale

saving culture medium and reagents

short screening cycle time

21

Coating CAb sample incubation HRP-IgG incubation TMB reaction stop & measurement

Illustrations of in-well miniature heterogeneous assay – ELISA


method for adding method for washing method for stopping reaction reagents and sample

pipetting

touch-loading

touch-loading

pipetting

touch-unloading

inverting to mix

touch-exchanging

pipetting

22

large surface area to volume ratio and short diffusion distance leading to the increase in initial binding velocity and short time to reach equilibrium

protein binding shown to obey single exponential kinetics

formation of antigen antibody complex shown single exponential behavior as well


1) Coating: 2 μg/ml GxR-IgG, 30 min

2) Reaction: 1:150K R-IgG-HRP

3) TMB reaction: 5min for IMAPlate and 15 min for

Nunc plate

1) Coating: 10 ng/ml GxR-IgG

2) Reaction: 1:20K R-IgG-HRP, 30min

3) TMB reaction: 15 min

single exponential

single exponential

+ steady state

single exponential

single exponential

+ steady state

single exponential

single exponential

+ steady state

1) Coating: IgG-HRP in 1 μg/ml BSA (diluted from 15

mg/ml BSA containing IgG-HRP)

2) TMB reaction: 5min for IMAPlate and 15 min for

Nunc plate

Time Course of

protein binding IgG binding complex formation

coating time (min)

0 10 20 30 40 50 60

Abso

rbance

/min

0

0.1

0.2

0.3

0.4

0.5

IMAPlate

MaxiSorp

reaction time (min)

0 10 20 30 40 50 60

Abso

rbance

/min

0

0.1

0.2

0.3

0.4

0.5

0.6

IMAPlate

Maxsorp

coating time (min)

0 10 20 30 40 50 60

Abso

rbance

0

0.2

0.4

0.6

0.8

1

1.2

1.4

IMAPlate

MaxiSorp

23

human IL-6 (pg/ml)

0 100 200 300 400 500 600

Ab

s4

50 -

Ab

s6

50

0

1

2

3

4

MaxiSorp 100ul

IMAPlate 5ul

human IL-17A (pg/ml)

0 200 400 600 800 1000

Abs4

50

- A

bs6

50

0

1

2

3

4

IMAPlate 20ul

MaxiSorp 100ul

In-well miniature ELISA

small sample volume

higher sensitivity

short time to result

less comsumption of reagents


DuoSet kit from R&D systems: 1) coating O.N. 2) 1st incubation 120min 3) 2nd incubation 120min 4) SA-HRP 20min and 5) TMB 20min.

Human IL-6 ELISA

from PerkinElmer Application Note: Using the EnSpire Multimode Plate Reader to Measure IMAPlate-Based Rapid Miniature ELISA for the Quantification of Troponin I

Rapid Miniature Troponin I ELISA IMAPlate: <3hrs incl. coating 96-well plate: 7hrs + ON coating

Human IL-17A ELISA

MAX™ Deluxe Kit from Biolegend: 1) coating O.N. 2) 1st incubation 120min 3) 2nd incubation 60min 4) SA-HRP 30min and 5) TMB 15min

24

Illustrations of in-well miniature heterogeneous assay – assays for compound screen

Advantages

easy wash procedure

solid phase enzyme assay

• able to eliminate the interference of colored compound on assay measurement

• possible to reuse the same coated plate several times when enzyme is stable

captive enzyme assay

• reducing other enzymes interference

• minimizing extract caused matrix effect for the assay


measurement enzyme activity enzyme activity labelled ligand

Compound screen with

solid phase captive enzyme compatitive

enzyme assay assay binding assay

Immobilization enzyme anti-enzyme antibody receptor

incubation compound crude extract compound and labelled ligand

25

Why switching from 2D cell culture to 3D cell culture?

Drug candidates showing promise in monolayer 2D cell cultures often fail in later stages of drug development.

cells altering the in vivo morphological and physiological characteristics when cultured in 2D

constrained cell signaling mechanisms between adjacent cells due to limited cell-cell interactions in 2D

3D spheroids having similar cell-cell interactions like tissues in vivo

3D spheroid displaying very similar gene expression pattern of clinical specimens


Illustration of hanging drop 3cell culture in Petri dish (limitations: medium change and spheroid transfer)

pipette cell suspension on the cover of Petri dish and culture the cells to form spheroids

wash off the spheroids transfer to 96-well plate

26

Illustrations of the preparation of spheroid micro-tissue and embryoid body in IMAPlateTM 5RC96

U87 spheroid in IMAPlateTM 5RC96 LN319 spheroid in assay plate HEK Spheroid

(2 days after seeding 5K, 2.5K and 1K cells: right to left) (2 days after exposure to compound) (expressing GFP)


methd for seeding cells method for medium change method for spheroid transfer

short incubation for addition of medium incubation and

cells to settle down medium change

Ф1.1 mm

27

Advantages of using IMAPlateTM 5RC96 as a “hanging drop” cell culture device for 3D cell culture


1. easy to change cell culture medium

2. no risk of loss spheroid when aspiring the medium from the non-capillarity reservoir

3. rapid spheroid transfer from IMAPlate to 96- & 384-well plate

4. no harsh operating procedure for spheroid transfer

5. able to prepare and test spheroids in the same IMAPlate

6. capable of preparing large quantity of spheroids

7. easy to estimate the size of spheroids

8. cells readily to form spheroids in small capillarity chamber

9. possible to use a shaker to facilitate the formation of spheroids

28

Summary

Features of IMAPlate

Miniature: minimizing sample consumption

reducing assay cost

Unique and flexible liquid handling simultaneous filling/emptying samples

retaining pipette liquid handling options

avoiding air gap formation with open end

Improvement for reaction and analysis increasing sensitivity for measurement

rapid adsorption and binding

short time for reaching equilibrium

UV-Vis-IR full spectrum measurement

Compatible with 96-well plate readers and automated liquid handling systems no special equipments required

Benefits of using IMAPlate

able to obtain multiparameter from limited amount of sample

minimizing the scale of upstream process such as cell culture, fermantation, and protein purification

no killing for small animal study and using less aninmal

able to follow the same aninmal at time points and reducing variation of individual difference

improving public images on animal research and industrial waste production (green technology)

manual high-throughput

short time to result

reducing time for screening cycle

enhancing lab productivity


NCL New Concept Lab GmbH Eichenstrasse 22, CH4313 Moehlin, Switzerland Tel: +41 61 853 08 20 / Fax: +41 61 853 08 23 Web: www.nclnewconceptlab.com

accompany you into the future


http://www.nclnewconceptlab.com/

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IMAPlateTM 5RC96 - world's first miniaturized analytical platform