• protocols used :Plasma activation, iRIf slide preparation

• 3 PDITC slides were prepared
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 2 h
• desiccator: 15 min
• stored at 4 °C

spotting pattern:
slides: 424, 012

slide: 254

Flush protocol:

Slide 012: Quotion picture

Slide 012: binding curves

Slide 424: Quotion picture

Slide 424: binding curves

Slide 254: Quotion picture

Slide 254: binding curves

→ the detection of GFP-, rabbit-, and mouse antibodies worked on all three slides

→ the increase in protein concentration of the spotted antigens on slide 254 does not lead to a increase of the detection signal → surface is with a protein concentration of 500 µg/ml already saturated

• protocols used :Plasma activation, iRIf slide preparation

• 1 PDITC slides were prepared
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 2 h
• desiccator: 15 min
• stored at 4 °C

spotting pattern:

slide-#:315

Flush protocol:

Slide 315: Quotion picture

Slide 315: binding curves

→ Binding of the anti-his antibody to the antigen (rhErbB2/Fc) was detected→ the (His tagged) antigen was immobilized on the surface

→ no binding of the affibody to its antigen couold be detected → either the affibody does not bind strong enough or the affibody is too small (~8 kDa) to generate a sufficient iRIf signal

• protocols used :Plasma activation, iRIf slide preparation

• 2 PDITC slides were prepared
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 2 h
• desiccator: 15 min
• stored at 4 °C

Spotting pattern:
Slides: 303,466

Flush protocol:

Slide 303: flushed with Serum (-) diluted 1:20 in BSA
Slide 466: flushed with Serum (-) diluted 1:30 in BSA

serum (-) 1:20 dilution:

Slide 303: Quotion picture

Slide 303: binding curves

→ strong binding to the salmonella antigen was detected → SIG 002 could also experienced a previous salmonella infection. Signal overall was quite weak.

serum (-) 1:30 dilution:

Slide 466: Quotion picture

Slide 466: binding curves

→the 1:30 diluted serum(-) shows little unspecific binding → weak binding to tetanus antigen could be caused by low anti tetanus antibody titer from previous vaccination

→overall weak signal, reuslts rather inconsistent→ use new stock of blood serum for previous experiments

• protocols used :Plasma activation, iRIf slide preparation

• 4 PDITC slides were prepared
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 2 h
• slides were sandwiched with NTA-solution and incubated o/n at 4 °C

• blocked slides with APTES blocking solution for 1 h
• washed slides 2x with PBS for 10 min
• incubated slides for 1 h in 1 % NiSO₄-solution
• washed slides with PBS/2x diluted PBS
• stored slides at 4 °C under N₂-atmosphere

• protocols used :Plasma activation, iRIf slide preparation

• 4 PDITC slides were prepared
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 2 h

spotting pattern:

slide-#: 208, 216, 423, 500

• spots were blocked 2x with 10 mg/mL BSA in PBS for 5 min
• slides were blocked with 10 mg/mL BSA in PBS for 30 min
• slides were washed with PBS 3x 10 min
• slides were washed with Aqua dest and dried

Flush protocol:

208: with PBS 423: with TBS 216: with TBS 500: with TBS (changed flush protocol: 1 ug/ml a-GFP; and used anti-Salmonella lysate 1:2 diluted in 5 mg/ml BSA in TBS)

PBS:

Slide 208: Quotion picture

Slide 208: binding curves

→ only very weak Salmonella antigen/antibody binding detectable, probably due to old salmonella antigen and antibody stocks

TBS:

Slide 216: Quotion picture

Slide 216: binding curves

Slide 423: Quotion picture

Slide 423: binding curves

→ only very weak or no Salmonella antigen/antibody binding detectable, probably due to old salmonella antigen and antibody stocks

Salmonella Lysate:

Slide 500: Quotion picture

Slide 500: binding curves

→ much unspecific binding detected, probably due to binding of E. coli proteins of the salmonella antibody lysate to other E. coli proteins still present in the purified mCherry and salmonella antigen (both were expressed in E. coli)

• protocols used :Plasma activation, iRIf slide preparation

• 4 PDITC slides were prepared
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 1 h
• PDITC incubation: 3 h
• slides were stored under N₂-atmosphere at 4 °C

• protocols used :Plasma activation, iRIf slide preparation

• 4 PDITC slides were prepared
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 1 h
• PDITC incubation: 3 h
• slides incubated o/n at 4 °C

• spotted Tetanus antigen

spotting pattern:

slide-#: 287, 024, 426, 443

• spots were blocked 2x with 10 mg/mL BSA in PBS for 5 min
• slides were blocked with 10 mg/mL BSA in PBS for 30 min
• slides were washed with PBS and 2x with 1/10 diluted PBS for 10 min
• slides were dried and measured in iRIf

Flush protocol:

Slides: 287, 024 (with Serum (-)) #287: Serum (-) was diluted only 1:10; changed to 1:30 because Serum(-) quite high Signal at negative control and Tetanus spot Flush protocol Slides: 426,443 (with Serum (+)) Flush protocol

Serum (-):

Slide 024: Quotion picture

Slide 024: binding curves

Slide 287: ROI selection

Slide 287: binding curves

→ slide 024 shows low unspecific binding due to 1:30 dilution of the serum(-), slide 287 shows high unspecific binding due to the 1:10 dilution of the serum(-)

→overall signal much weaker than before, probably due to repeated freezing of the blood serum

Serum (+):

Slide 426: ROI selection

Slide 426: binding curves

Slide 433: ROI selection

Slide 433: binding curves

→slide 426 shows relativly weak unspecific binding and moderate binding of proteins to the Tetanus antigen, slide 433 has more unspecific binding

→overall signal much weaker than before, probably due to repeated freezing of the blood serum

• protocols used :Plasma activation, iRIf slide preparation

• 2 PDITC slides were prepared
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 1 h
• PDITC incubation: 3 h
• slides were sancwiched with NTA-solution and incubated o/n at 4 °C

• blocked slides with APTES blocking solution for 1 h
• washed slides 2x with PBS for 10 min
• incubated slides for 1 h in 1 % NiSO₄-solution
• washed slides with PBS/2x diluted PBS
• stored slides at 4 °C under N₂-atmosphere

• spotted 3 µL of affibody (from Stockholm) and controls (GFP, bBSA)

spotting pattern:

slide-#: 500

→ because of the His tag of the antigen it binds to the Ni-NTA surface→no specific antigen/affibody binding was detectable
→ retry the experiment with immobilized antigen on a PDITC surface, flush the affibody through the flow camber

• protocols used :Plasma activation, PDMS-PDITC, DNA + Cellfree Expression on PDMS

• prepare 3 PDMS slides for spotting with DNA and cell-free expression

• used 2 flow cells made on 18.08.2015: one with same pattern as flow cells used in iRIf (conic, one hole) and one with three holes and cavities, +1 made on 16.08.15 with Standard flow cell form and small borders
• washed PDMS slides with Ethanol and H₂O
• activated slides in plasma generator with 30 L/h for 1 min
• incubated slides in APTES o/n at room temperature

• Flow cells were washed individually with ethanol
• Washing step of 5 min in ethanol in slideholder
• Flow cells were dried with wafergun
• heated 90 min in oven at 70°C
• cooled down with wafergun
• Flow cells were incubated in PDITC for 2h
• filled slideholder with EtOH and discarded it immediately
• washed 2x 5 min with EtOH in slideholder
• filled slideholder again with EtOH and discarded it immediately
• dried with wafergun
• stored at 4°C in nitrogen atmosphere

• spots of DNA were put on flow cells according to the following pattern
• incubation overnight at room temperature in humid atmosphere

spotting pattern (slide 61a):

spotting pattern (slide 61b):

spotting pattern (slide 61c):

• checked for Cy3-intensity by spotting a 1.5 µl spot of Cy3-labeled GFP (33 ng/µl) on old PDMS flow cell from 18.08.2015
• spot was let dry in at room temperature until its borders were still visible
• checked for fluorescence in Mircoarray Scanner

• Cy3 spot could be detected

slide 254: Retikulozyte, slide 012: Roth, slide 423: Koko

• protocols used :Plasma activation, PDMS-PDITC, DNA + Cellfree Expression on PDMS

• prepare 2 PDMS slides for spotting with DNA

• used 2 flow cells made on 18.08.2015: one with same pattern as flow cells used in iRIf (conic, one hole) and one with three holes and cavities
• washed PDMS slides with Ethanol and H₂O
• activated slides in plasma generator with 30 L/h for 1 min
• incubated slides in APTES o/n at room temperature

• each slide was washed with ethanol
• a 5 min washing step in Ethanol followed
• after drying with wafergun slides were baked in oven (old one) for 120 min at 70°C
• slides were cooled down with wafergun
• incubation in PDITC for 3h30 at RT
• filled slideholder with EtOH and discarded it immediately
• washed 2x 5 min with EtOH in slideholder
• filled slideholder again with EtOH and discarded it immediately
• dried with wafergun
• spots of DNA were put on flow cells according to the following pattern
• incubation overnight at room temperature in humid atmosphere

spotting pattern (slide 60a):

spotting pattern (slide 60b):

• spots were let dry in at 60°C for 30 min still in humid atmosphere
• expression was effected with 15 µl (60b)/7 µl (60a each flow cell) of Koko mix for 2h at 37°C
• flow cells were then checked for fluorescence with microscope of AG Eimer

• no fluorescence could be observed

60a

60b

• after storage at 4°C in humid atmosphere overnight flow cells were again checked for fluorescence with microscope of AG Eimer
• no fluorescence could be observed

60a

60b

• protocols used :Plasma activation, iRIf slide preparation

• prepared 3 PDITC slides
• Plasma activation: 40 l/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 3 h

• slides were sandwiched with NTA-solution and incubated o/n at 4 °C

• slides were blocked with APTES blocking solution for 1 h
• slides were washed 2x with PBS
• slides were incubated with NiSO₄-solution for 1 h and 20 min
• slides were washed with PBS/2x diluted PBS

• cell free GFP Expression mix was spotted on slides (15 µL per spot)and incubated it 3 h at 37 °C
• spotted off- slide cell free expressed GFP on the two slides after the first incubation and incubated o/n at 4°C

spotting pattern:

slide-#: 303,466

• blocked spots 2x with 10 mg/mL BSA in PBS for 5 min
• blocked slides with 10 mg/mL BSA in PBS for 30 min
• washed slides with PBS/diluted PBS + 20 mM imidazole/diluted PBS

Flush protocol: slide 466 and 303

ROI selection of slide 466

picture of slide 466 from the microarray scanner (exitation at 635 nm

binding curves of slide 466

→ salt deposits on the surface of slide 303 made an evaluation difficult

• protocols used :Plasma activation, iRIf slide preparation

• prepared 3 PDITC slides
• Plasma activation: 40 l/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 3 h

• prepared 3 Ni-NTA slides according to iRIf slide preparation protocol
• PDITC slides were incubated o/n with NTA at 4°C
• slides were blocked in APTES blocking solution for 30 min
• slides were incubated in NiSO4 for 1 h
• slides were washed with PBS/diluted PBS
• slides were stored at 4 °C

• spotted free expression mix on two slides (429, 424) and incubated it 3 h at 37 °C
• spotting mask was accidently removed after incubation –> spotted Solutions probably mixed (no definite spots)
• spotted off- slide cell free expressed GFP on the two slides after the first incubation and incubated o/n at 4°C
• spotted 1 slide (500) with only off-slide cell-free expressed GFP and incubated o/n at 4°C

spotting pattern:

slide-#: 429, 424

spotting pattern:

slide-#: 500

• spots were blocked 2x with 10 mg/mL BSA for 5 min
• slides were blocked with 10 mg/mL BSA for 30 min
• slides were washed with PBS/diluted PBS + 20 mM imidazole/diluted PBS

slide 424 and 500

429 was not measured because the other slides showed that GFP was expressed, so the Experiment will be repeated and the above mentioned mistake (removal of spotting masks after 37 °C incubation) will be avoided.

Only slide 500 was evaluated. Slide 424 and 429 showed very bad surface during measurements which was wiped off → Difficult to evaluate Slide 500: spots 4 and 8 had to be excluded from evaluation.

Slide 500: ROI selection

Slide 500: Binding curves

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 PDITC slides (208, 216)

• prepared 2 PDITC slides
• Plasma activation: 40 l/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 3 h

• prepared 2 Ni-NTA slides according to iRIf slide preparation protocol
• PDITC slides were incubated o/n with NTA at 4°C
• slides were blocked in APTES blocking solution for 30 min
• slides were incubated in NiSO4 for 1 h
• slides were washed with PBS/diluted PBS
• slides were stored at 4 °C

• spotted with cell-free expressed Tetanus samples, GFP and bBSA

spotting pattern:

slide-#: 216

slide-#: 208

• spots were blocked 2x with 10 mg/mL BSA for 5 min
• slides were blocked with 10 mg/mL BSA for 30 min
• slides were washed with PBS/diluted PBS + 20 mM imidazole/diluted PBS

Slide 208: Only 5 spots (spot 3, 5, 7, 8 and 9) could be evaluated.

Slide 208: ROI selection

Slide 216: ROI selection

Slide 208: Binding curves

Slide 216: Binding curves

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 PDITC slides

• prepared 2 PDITC slides
• Plasma activation: 40 l/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 3 h
• spotted 3 µL of Antigen and controls (GFP)

spotting pattern:

slide-#: 287, 504

• spots were blocked 2x with 10 mg/mL BSA in PBS for 5 min
• slides were blocked with 10 mg/mL BSA in PBS for 30 min
• slides were washed with Aqua dest and dried

Flush protocol slide 287

no Strep-Cy5 was flushed over slide –> no scanner Pictures

Flush protocol slide 504

Slide 287: Selection of ROI

Slide 287: Binding curves

Slide 504: Selection of ROI

Slide 504: Binding curves

→ detection of Tetanus antibodys from blood serum after vaccination is possible
→ huge cross reactivity with bBSA spot→probably due to biodinidase enzyme

• Check if antibodies in blood of freshly vaccinated Patient SIG002 binds to our self-expressed Tetanus Antigen
• detection of antibodies in blood with anti-human (mouse)
• detection of anti-human with HRP-labeled anti-mouse

• 100 µL of each Tet-Tox and bBSA concentration were pipetted into maxisorb-plate

• 8:34 am: all wells exept H1 were blocked with 5 mg/mL BSA in PBS
• wells were washed with PBS 2 times for ~2 min
• 10:00 am: 100 µL of 1/10 diluted blood Serum were added to well A1-H1 and again 100 µL of 5 mg/mL BSA in PBS to A2-H2 and A3-H3
• all solutions were overlayed with 100 µL PBS
• wells were washed with PBS 3 times for ~2 min
• 11.07 am: 100 µL of anti-human (1 µg/mL) were added to well A1-H1 and again 100 µL of 5 mg/mL BSA in PBS to A2-H2 and A3-H3
• all solutions were overlayed with 100 µL PBS
• wells were washed with PBS 3 times for ~2 min
• 1:00 pm: 100 µL of HRP-anti-mouse (1 µg/mL) were added to well A1-H1 and 100 µL of HRP-Strep (0.5 µg/mL) to A2-H2 and A3-H3
• all solutions were overlayed with 100 µL PBS
• wells were washed with PBS 5 times for ~2 min

• Hydrogenperoxide and 3,3´,5,5´-Tetramethylbenzidine were added to each well and the Absorption was measured in the plate reader

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 PDITC iRif slides
• slide#:

• slides were washed according to iRIf slide wash steps
• Plasma activation: 40 L/h 5 min
• APTES incubation: 30 min
• PDITC incubation 3.5 h

• slides were blocked in APTES blocking solution for 45 min
• slides were incubated in NiSO₄ for 1 h
• slides were washed with PBS/diluted PBS
• slides were stored at 4 °C

* spotted 3 µL of Salmonella desalt and lysate and controls (GFP-Lysate, GFP-desalt, bBSA)

spotting pattern:

slide-#: 012, 426

• spots were blocked 2x with 10 mg/mL BSA in TBS for 5 min
• slides were blocked with 10 mg/mL BSA in TBS for 30 min
• slides were washed with TBS/diluted TBS + 20 mM imidazole/diluted TBS

• protocols used :Plasma activation, iRIf slide preparation

• prepare 4 PDITC iRif slides
• slide#: 423, 215, 254, 024

• slides were washed according to iRIf slide wash steps
• Plasma activation: 40 L/h 5 min
• APTES incubation: 30 min
• PDITC incubation 3.5 h

• slides were blocked in APTES blocking solution for 45 min
• slides were incubated in NiSO₄ for 1 h
• slides were washed with PBS/diluted PBS
• spotted 3 µL of cell free expressed GFPs and controls (GFP-Lysate, GFP-desalt, bBSA)

spotting pattern:

slide-#: 423, 254

slide-#: 024, 215

• spots were blocked 2x with 10 mg/mL BSA for 5 min
• slides were blocked with 10 mg/mL BSA for 30 min
• slides were washed with PBS/diluted PBS + 20 mM Imidazole/diluted PBS

slide 215: old Setup

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 PDITC iRif slides
• slide#: 466, 253

• slides were washed according to iRIf slide wash steps
• Plasma activation: 40 L/h 5 min
• APTES incubation: 30 min
• PDITC incubation 3.5 h

• slides were blocked in APTES blocking solution for 45 min
• slides were incubated in NiSO₄ for 1 h
• slides were washed with PBS/diluted PBS
• two slides (466, 253) were spotted with 15 µl per spot spottingmasks

Spotting:

• slides were covered with glass slides to avoid evaporation
• slides were incubated for for 3 h at 37 °C
• measurement in iRIf

Slide 253: Quotion picture

Slide 466: Quotion picture

→ no cell free expressed GFP could be detected
→ on slide 253 even the spotted GFP lysate was not detectable, this is probably due to repeaded problems with the correct flooding of the flow cell during the measurement with the iRIf setup
→ the experiment will be repeated with additional spots of „off slide“ cell free expressed GFP

• prepare 2 PDITC iRif slides
• slide#: 315, 502

• slides were washed according to iRIf slide wash steps
• Plasma activation: 40 L/h 5 min
• APTES incubation: 30 min
• PDITC incubation 3.5 h
• slides were washed and dried with wafer gun
• 3 µL of cell free expressed GFP, GFP Lysate, GFP desalt, bBSA, BSA and mCherry were spotted.

spotting pattern:

slides were measured with Serum(-) and Serum(+)

• blocked spots 2x with 10 mg/mL BSA for 5 min
• blocked slides with 10 mg/mL BSA for 30 min
• washed slides with PBS/diluted PB + 20 mM Imidazole/diluted PBS for 10 min

Flush protocol Measuring with Serum (-) [not immunized] and Serum (+) [immunized]/a-GFP:

Serum (-) / Serum (+)

Slide 315: Selection of ROIs

Slide 315: Binding curves

Slide 502: Selection of ROIs

Slide 502: Binding curves

• protocols used :Plasma activation, PDMS-PDITC, DNA + Cellfree Expression on PDMS

• prepare 2 PDMS slides for spotting with DNA

• used 2 flow cells made on 16.08.2015 with same pattern as flow cells used in iRIf (conic, one hole, one with pillars/one bad)
• washed PDMS slides with Ethanol and H₂O
• activated slides in plasma generator with 30 L/h for 1 min
• incubated slides in APTES o/n at room temperature

• each slide was washed with ethanol
• a 5 min washing step in Ethanol followed
• after drying with wafergun slides were baked in oven (better one) for 90 min at 70°C
• slides were cooled down with wafergun
• incubation in PDITC for 2h at RT
• filled slideholder with EtOH and discarded it immediately
• washed 2x 5 min with EtOH in slideholder
• filled slideholder again with EtOH and discarded it immediately
• dried with wafergun
• flow cells were spotted with 1 µl spots and incubated over night at room temperature in humid atmosphere

spotting pattern:

• expression was effected with 15 µl of Koko mix for 2h at 37°C
• flow cells were then checked for fluorescence with microscope of AG Eimer

• for both flow cells no fluorescence could be observed

56a

56b

• flow cells were disassembled and washed with PBS and H₂O
• dried with wafergun
• expression was repeated with 15 µl of EasyExpress-Mix (AG Roth) at 37°C in humid atmosphere
• fluorescence was measured after 1h and 2h with microscope of AG Eimer

• a few fluorescent spots could be observed

56a after 1h

56a after 2h

56b after 1h

56b after 2h

• protocols used :Plasma activation, PDMS-PDITC, DNA + Cellfree Expression on PDMS

• prepare 2 PDMS slides for spotting with DNA

• used 2 flow cells (one with same pattern as flow cells used in iRIf and pillars, one with cavities and three lines)
• washed PDMS slides with Ethanol and H₂O
• used correct APTES solution (1 mL APTES, 1 mL H₂O in 18 mL Ethanol)
• activated slides in Plasma Generator with 40 L/h for 1 min
• incubated slides in APTES o/n

• each slide was washed with ethanol
• a 5 min washing step in Ethanol followed
• after drying with wafergun slides were baked in oven (better one) for 90 min at 70°C
• slides were cooled down with wafergun
• incubation in PDITC for 3h at RT
• filled slideholder with EtOH and discarded it immediately
• washed 2x 5 min with EtOH in slideholder
• filled slideholder again with EtOH and discarded it immediately
• dried with wafergun
• cells were spotted with 3 µl spots and incubated over night at room temperature in humid atmosphere
• sample 1 was diluted 1:2 (original oncentration 44,8 ng/µl) in NaPi buffer 150 mM

spotting pattern:

flow cell 55a

flow cell 55b

• Petri dish with flow cells was put in oven at 60°C for 30 min
• Flow cells were then washed with aqua dest. and dried with wafergun
• Expression was effected for 20, 40 and 130 min with AG Roth expression mix at 37°C (not sealed, no humid atmosphere)
• flow cells were checked for fluorescence in microscope of AG Eimer
• afterwards flow cells were washed with 2 ml PBS and aqua dest. and thene dried with wafergun
• stored in petri dish sealed with parafilm at 4°C

• Expression of our GFP conctruct and the control construct could be observed on both flow cells
• Expressed GFP seems to diffuse with the time. This might be due to the transport of the slides from the incubator to the microscope and back.

55a after 20 min incubation time

55a after 40 min incubation time

55a after 130 min incubation time

55b after 20 min incubation time

• Expression was effected for 2h with Koko expression mix at 37°C (sealed, humid atmosphere)
• flow cells were checked for fluorescence in microscope of AG Eimer
• afterwards flow cells were washed with 2 ml PBS and aqua dest. and thene dried with wafergun
• stored in petri dish sealed with parafilm at 4°C

• no fluorescence could be observed for both flow cells as well as for Tobis 4 flow cells

55a after 2h

55b after 2h

• protocols used :Plasma activation, iRIf slide preparation

• prepare 4 PDITC iRIf slides
• slides : 504, 500, 216, 208

• 4 PDITC iRIf slides were prepared
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 2 h

• slides were sandwiched with 60 µL NTA-solution and incubated o/n at 4 °C

• slides were blocked in APTES blocking solution for 1 h
• slides were incubated in NiSO<Sub>4</Sub> for 1 h
• slides were washed with PBS/diluted PBS
• 3 µL of cell free expressed GFP, GFP Lysate, GFP desalt, bBSA, BSA and mCherry were spotted.

spotting pattern:

3 of the slides (208, 216, 504) were measured with Serum(-) and Serum(+)
1 of the slides (500) was measured with Serum(-) and anti-GFP
(slide from Exp. 50c was spotted similar and also measured with Serum(-) and anti-GFP)

• blocked spots 2x with 10 mg/mL BSA for 5 min
• blocked slides with 10 mg/mL BSA for 30 min
• washed slides with PBS/diluted PB + 20 mM Imidazole/diluted PBS for 10 min

Flush protocol Measuring with Serum (-) [not immunized] and Serum (+) [immunized]/a-GFP:

Serum (-) / Serum (+)

Slide 208: Selection of ROIs

Slide 208: Binding curves

Slide 502: Selection of ROIs

Slide 502: Binding curves

Serum (-) / a-GFP

Slide 424: Selection of ROIs

Slide 424: Binding curves

The relative intensity shift for the three approaches (Serum(-)Serum(+) on Ni-NTA ; Serum(-)Serum(+) on PDITC; Serum(-)a-GFP on Ni-NTA) were compared. As expected and seen in the binding curves cellfree expression Mix (His-GFP) shows no signal on PDITC (other proteins within the mix bind to the surface) but on specific Ni-NTA surface. Taken amount of serum performs less (2-3 fold) than 3 ug/ml a-GFP.

Evaluation of the relative intensity shift. From left to right spot 1-11. For each spot rel. intensity shift is plotted with bars for Serum(-)flush and Serum(+)/a-GFP

Emission within 532nm channel (mCherry) after iRIf measurement. Averaged over all samples.

Emission within 635nm channel (Cy5) after iRIf measurement. Averaged over all samples

• protocols used :Plasma activation, iRIf slide preparation

• prepare 1 PDITC iRIf slides
• slides : 467

• 1 PDITC iRIf slides were prepared
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 30 min
• PDITC incubation: 2 h

• 1 slide was spotted alternating with bBSA and BSA for measurement in own device, in total 14 spots

• slide for own device was blocked (2x 10 mg/mL BSA on spots for 5 min, 1x in 10 mg/mL BSA for 30 min)

• protocols used :Plasma activation, Halosurface from AG Piehler

• second try with halo surface

• activated slides 253 and 254 with 40 L/h for 5 min
• cross-sandwiched slides with 15 µL PLL-PEG-HTL
• incubated slides for 10 min
• washed slides with Aqua dest and stored them in slideholder o/n (no N₂-atmosphere)

• blocked slides in 10 mg/ml BSA for 1h
• washed slides with aqua dest. and dried with nitrogengun
• spotted slides and incubated for 1 h at roomtemperature

Spotting:

• blocked wells twice with 10 mg/ml BSA for 5 min
• blocked slides 30 min in 10 mg/ml BSA
• washed slides with water and dried with nitrogengun
• measured fluorescence intensity with fluorescence microscope
• measurement in iRIf

• protocols used :Plasma activation, Halosurface from ABCR

• second try with halo surface

• activated slides 012 and 215 with 40 L/h for 5 min
• sandwiched slides with 80 µL abcr-silane
• incubated slides for 1 h
• washed slides with Aqua dest and stored them in slideholder o/n (no N₂-atmosphere)

• blocked slides in 10 mg/ml BSA for 1h
• washed slides with aqua dest. and dried with nitrogengun
• spotted slides and incubated for 1 h at roomtemperature

Spotting:

• blocked wells twice with 10 mg/ml BSA for 5 min
• blocked slides 30 min in 10 mg/ml BSA
• washed slides with water and dried with nitrogengun
• measured fluorescence intensity with fluorescence microscope
• measurement in iRIf

• protocols used :Plasma activation, iRIf slide preparation

• washed 1 iRIf slides
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 45 min
• PDITC incubation: 2 h
• stored slides at 4 °C o/n

• slide was spotted alternating with bBSA and BSA for measurement in own device, in total 14 spots

• slide was blocked (2x 10 mg/mL BSA on spots for 5 min, 1x in 10 mg/mL BSA for 30 min)

• protocols used :Plasma activation, iRIf slide preparation, Halosurface from Wiesmüller

• washed 2 iRIf slides
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 45 min
• PDITC incubation: 2 h
• stored slides at 4 °C o/n

• slide 423 and 287 were incubated with Wiesmüller Halo-Linker 2 o/n in slideholder

• washed slides with Aqua dest. and dried with nitrogengun
• blocked slides in 10 mg/ml BSA for 1h
• washed slides with aqua dest. and dried with nitrogengun
• spotted slides and incubated for 1 h at roomtemperature

Spotting:

• blocked wells twice with 10 mg/ml BSA for 5 min
• blocked slides 30 min in 10 mg/ml BSA
• washed slides with water and dried with nitrogengun
• measured fluorescence intensity with fluorescence microscope
• measurement in iRIf

• protocols used :Plasma activation, iRIf slide preparation, Halosurface from Wiesmüller

• washed 2 iRIf slides
• Plasma activation: 40 L/h, 5 min
• APTES incubation: 45 min
• PDITC incubation: 2 h
• stored slides at 4 °C o/n

• slide 426 and 466 were incubated with Wiesmüller Halo-Linker 1 o/n in slideholder

• washed slides with Aqua dest. and dried with nitrogengun
• blocked slides in 10 mg/ml BSA for 1h
• washed slides with aqua dest. and dried with nitrogengun
• spotted slides and incubated for 1 h at roomtemperature

Spotting:

• blocked wells twice with 10 mg/ml BSA for 5 min
• blocked slides 30 min in 10 mg/ml BSA
• washed slides with water and dried with nitrogengun
• measured fluorescence intensity with fluorescence microscope
• measurement in iRIf

• protocols used :Plasma activation, iRIf slide preparation

• test if antibody binding is detectable with self built iRIf setup

• used two already prepared iRIf PDITC slides from Exp. 47b (265) and 49c (503)
• spotted bBSA (200 µg/ml) and BSA (10 mg/ml) in an alternating pattern on slide 503
• spotted anti HCV antibodys (rabbit, polyclonal, 500 µg/ml)and BSA (10 mg/ml) an alternating pattern on slide 265
• spotted 18 spots in total on each slide

• protocols used : PDMS-PDITC

• repeat immobilization of Cy5-labeled DNA on PDMS slides with correct APTES solution

• used 2 flow chamber slides with same pattern as flow Chambers used in iRIf (conic, one hole)
• washed PDMS slides with Ethanol and H₂O
• used correct APTES solution (1 mL APTES, 1 mL H₂O in 18 mL Ethanol)
• activated slides in Plasma Generator with 40 L/h for 1 min
• incubated slides in APTES o/n

• each slide was washed with ethanol
• a 5 min washing step in Ethanol followed
• after drying with wafergun slides were baked in oven for 90 min at 70°C
• slides were cooled down with wafergun
• incubation in PDITC for 5h40min at RT
• filled slideholder with EtOH and discarded it immediately
• washed 2x 5 min with EtOH in slideholder
• filled slideholder again with EtOH and discarded it immediately
• dried with wafergun
• 1 slide (51a) was spotted with 1 µl spots and incubated over night
• sample 1 was diluted 1:4,64 (original oncentration 116 ng/µl) in NaPi buffer 150 mM

spotting pattern 51a:

• slide (b) was spotted with 3 µl spots and incubated for 2 nights at room temperature
• sample 1 was diluted 1:4,64 (original concentration 116 ng/µl) in NaPi buffer 150 mM

spotting pattern 51b:

• DNA was let dry in
• slide 51a was washed with H₂O and dried with wafergun

• slide 51a was measured in Microarray Scanner for Cy5
• no spots could be seen

• DNA was let dry in
• slide 51b was washed with H₂O and dried with wafergun

• measured in Microarray Scanner for Cy5
• 3 spots could be (hardly) seen with the eye. The intensity was too low to be visible on the derived image file. Background was approximately at 0.7 to 1.7. The spot was between 1.2 and 4.0.

• 15 µl of cell-free expression mix (EasyExpress (Kubick) of AG Roth) were added to each flow cell
• incubation for 20 min at 37°C
• fluorescence was measured in microscope of AG Eimer
• no expressed GFP could be detected

• protocols used :Plasma activation, iRIf slide preparation

• prepare 1 Ni-NTA slides for measurement in iRIf
• slide-#: 424

• washed 1 iRIf slides
• Plasma activation: 80 L/h, 5 min
• APTES incubation: 1 h
• PDITC incubation: 5 h
• stored slides at 4 °C o/n

• sandwiched slides with 60 µL NTA-solution per sandwich and incubated o/n at 4 °C

• blocked slides in APTES/PDITC blocking solution for 1 h
• washed 2x with PBS
• incubated slides for 1h 15 min in NiSO<Sub>4</Sub>-solution
• washed slides 2x with PBS
• washed slides with 1/10 diluted PBS
• stored slides for later use under N₂-atmosphere at 4 °C

• 3 µL of cell free expressed GFP, GFP Lysate, GFP desalt, bBSA, BSA and mCherry were spotted.

spotting pattern:

slide (424) was measured with Serum(-) and anti-GFP (measured together with slides from Exp. 54b)

• blocked spots 2x with 10 mg/mL BSA for 5 min
• blocked slides with 10 mg/mL BSA for 30 min
• washed slides with PBS/diluted PB + 20 mM Imidazole/diluted PBS for 10 min

Flush protocol Measuring with Serum (-) [not immunized] and Serum (+) [immunized]/a-GFP:

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 Ni-NTA slides for measurement in iRIf
• Changed buffer: use always TBS instead of PBS (because TBS is Elution buffer)
• also use TBS for iRIf measurement

• washed 2 iRIf slides
• Plasma activation: 80 L/h, 5 min
• APTES incubation: 1 h
• PDITC incubation: 5 h
• stored slides at 4 °C o/n

• sandwiched slides with 60 µL NTA-solution per sandwich and incubated o/n at 4 °C

• blocked slides in APTES/PDITC blocking solution for 1 h
• washed 2x with PBS
• incubated slides for 1h 15 min in NiSO₄ -solution
• washed slides 2x with PBS
• washed slides with 1/10 diluted PBS
• spotted antigens were diluted in TBS
• spotted slides 500 and 501

Spotting:

• spots were blocked 2x with 10 mg/mL BSA in TBS for 5 min
• slides were blocked with 10 mg/mL BSA in TBS for 30 min
• slides were washed with TBS
• slides were washed with 1/10 diluted TBS + 20 mM imidazole
• slides were washed with 1/10 diluted TBS

slide 500 was measured in new setup
slide 501 could not be measured, air bubbles appeared every time the slide was flushed –> surface must be really hydrophobic. Maybe slide was destroyed during tests with PDMS-stamps.

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 Ni-NTA slides for measurement in iRIf
• Changed buffer: use always TBS instead of PBS (because TBS is Elution buffer)
• also use TBS for iRIf measurement

• washed 2 iRIf slides
• Plasma activation: 80 L/h, 5 min
• APTES incubation: 1 h
• PDITC incubation: 5 h
• stored slides at 4 °C o/n

• sandwiched slides with 60 µL NTA-solution per sandwich and incubated o/n at 4 °C

• blocked slides in APTES/PDITC blocking solution for 1 h
• washed 2x with PBS
• incubated slides for 1h 15 min in NiSO₄-solution
• washed slides 2x with PBS
• washed slides with 1/10 diluted PBS
• antigenes were diluted in TBS
• spotted slide 216 and 467

Spotting:

• spots were blocked 2x with 10 mg/mL BSA in PBS for 5 min
• slides were blocked with 10 mg/mL BSA in TBS for 30 min
• slides were washed with TBS
• slides were washed with 1/10 diluted TBS + 20 mM imidazole
• slides were washed with 1/10 diluted TBS

216 was measured in old Setup´
467 was measured in new Setup

Slide 24: Selection of ROIs: 1 HIV spot (spot 2) and one HCV spot (spot 3) were excluded due to high amounts of salt on it

Slide 24: Binding curves

Slide 502: Selection of ROIs

Slide 502: Binding curves for all spots

Slide 502: Binding curve (second Salmonella spot excluded)

Results: We have specific antibody/antigen binding for Salmonella! (a-HIV/HCV do not bind…) Slide 502: During measurement within the first buffer step/blocking step most of the second Salmonella spot was washed away. Related to this the spot behaves strange. Slide 24: Also anti-salmonella lysate was tested. Here we see (as expected) stronger unspecific binding at other spots (nevertheless greatest binding for the salmonella spots). The unspecific/transient binding proteins get mostly washed away during the following buffer step.

Slide 24: Quotient picture (before a-Salmonella / after a-Salmonella + a-GFP)

Slide 502: Quotient picture (before a-Salmonella / after a-Salmonella + a-GFP)

• protocols used :Plasma activation, iRIf slide preparation

• prepare 1 PDITC slides for measurement in iRIf

• washed 1 iRIf slides
• Plasma activation: 80 L/h, 5 min
• APTES incubation: 1 h
• PDITC incubation: 5 h
• stored slides at 4 °C under N₂-atmosphere

• used slides for measurement in own device –> Exp. 52

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 PDITC slides for measurement in iRIf
• Changed buffer: use always TBS instead of PBS (because TBS is Elution buffer)
• also use TBS for iRIf measurement

• washed 2 iRIf slides
• Plasma activation: 80 L/h, 5 min
• APTES incubation: 1 h
• PDITC incubation: 5 h
• stored slides at 4 °C under N₂-atmosphere

• spotted slides 502 and 024
• incubated at 4°C o/n

Spotting:

• spots were blocked 2x with 10 mg/mL BSA in PBS (should have used TBS) for 5 min
• slides were blocked with 10 mg/mL BSA in PBS for 30 min

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 PDITC slides for measurement in iRIf

• washed iRIf slides
• Plasma activation: 80 L/h, 5 min
• APTES incubation: 1 h
• PDITC incubation: 5 h
• spotted 2 slides (426, 253) with bBSA/BSA for new device

• blocked spots 2x with 10 mg/mL BSA for 5 min
• blocked slides with 10 mg/mL BSA for 30 min
• washed slides with ddH₂O and dried with wafer gun

• protocols used : PDMS-PDITC, Plasma activation

• immobilize Cy5-labeled DNA on PDMS slides

• used 2 flow chamber slides with 3 holes
• washed PDMS slides with Ethanol and H₂O
• used just 200 µL APTES in Acetone for APTES solution instead of 1 mL APTES in Ethanol
• activated slides in Plasma Generator with 40 L/h for 1 min
• incubated slides in APTES solution for 2 days+

• each slide was washed with ethanol
• a 5 min washing step in Ethanol followed
• after drying with wafergun slides were baked in oven for 90 min at 70°C
• slides were cooled down with wafergun
• incubation in PDITC for 5h40min at RT
• filled slideholder with EtOH and discarded it immediately
• washed 2x 5 min with EtOH in slideholder
• filled slideholder again with EtOH and discarded it immediately
• dried with wafergun
• 1 slide (a) was spotted with 1 µl spots and incubated over night
• sample 1 was diluted 1:4,64 (original oncentration 116 ng/µl) in NaPi buffer 150 mM

spotting pattern 48a:

• slide (b) was spotted with 3 µl spots and incubated for 2 nights at room temperature
• sample 1 was diluted 1:4,64 (original concentration 116 ng/µl) in NaPi buffer 150 mM

spotting pattern 48b:

• DNA was let dry in
• slide 48a was washed with H₂O and dried with wafergun

• slide48a was measured in Microarray Scanner for Cy5
• no spots could be seen

• DNA was let dry in
• slide 48b was washed with H₂O and dried with wafergun

• slide48b was measured in Microarray Scanner for Cy5
• no spots could be seen

• protocols used :Plasma activation, iRIf slide preparation

• prepare 1 PDITC iRIf slides for measurement of antigens, PhyA or bBSA in own device

• prepared 1 PDITC slides
• plasma activation: 80 L/h
• APTES incubation: 30 min
• PDITC incubation: 2 h
• slides were stored in slide holder under N₂-atmosphere

• used slides for measurement in own device –> Exp. 52

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 PDITC iRIf slides for measurement of antigens, PhyA or bBSA in own device

• prepared 2 PDITC slides
• plasma activation: 80 L/h
• APTES incubation: 30 min
• PDITC incubation: 2 h
• slides were stored in slide holder under N₂-atmosphere

• spotted 3 µL of different PhyAs and controls on 2 slides

Spotting:

• spots were blocked 2x with 10 mg/mL BSA for 5 min
• sildes were blocked with 10 mg/mL BSA for 30 min

slide 012 was measured, the other slide is broken and could not be measured.

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 Ni-NTA slides for measurement of cell free expressed Proteins (GFP/tYFP) or antigen lysate

• prepared 2 PDITC slides
• plasma activation: 80 L/h
• APTES incubation: 45 min
• PDITC incubation: 2h
• slides were sandwiched with NTA solution, 1 slide was sandwiched with a normal glass slide

• slides were incubated in blocking solution for 1 h
• slides were incubated in Nickel sulfate solution for 1 h
• slides were washed 2x in PBS and 1x in diluted PBS
• slides were dried and stored under N₂-atmosphere

• spotted slides 303 and 429 with samples from cell-free
• incubated at 4°C o/n

Spotting (Slide 303):

Spotting (Slide 429):

• protocols used :Plasma activation, iRIf slide preparation

• prepare 1 Ni-NTA slides for measurement of cell free expressed Proteins (GFP/tYFP) or antigen lysate

• prepared 1 PDITC slides
• plasma activation: 80 L/h
• APTES incubation: 45 min
• PDITC incubation: 2h
• slides were sandwiched with NTA solution, 1 slide was sandwiched with a normal glass slide

• slides were incubated in blocking solution for 1 h
• slides were incubated in Nickel sulfate solution for 1 h
• slides were washed 2x in PBS and 1x in diluted PBS
• slides were dried and stored under N₂-atmosphere

• spotted cell-free expressed GFP and His-Lysate on slide 208
• incubation o/n at 4 °C

Spotting:

• spots were blocked 2x with 10 mg/mL BSA in PBS for 5 min
• slides were blocked with 10 mg/mL BSA in PBS for 30 min
• slides were washed in PBS for 10 min
• slides were washed in d1/10 diluted PBS with 20 mM Imidazole
• slides were washed in d1/10 diluted PBS

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 Ni-NTA slides for measurement of cell free expressed Proteins (GFP/tYFP) or antigen lysate

• prepared 2 PDITC slides
• plasma activation: 80 L/h
• APTES incubation: 45 min
• PDITC incubation: 2h
• slides were sandwiched with NTA solution, 1 slide was sandwiched with a normal glass slide

• slides were incubated in blocking solution for 1 h
• slides were incubated in Nickel sulfate solution for 1 h
• slides were washed 2x in PBS and 1x in diluted PBS
• slides were dried and stored under N₂-atmosphere

• spotted slide 254 and 215 with cell free expressed proteins
• 3 µL of cell free expressed His-tYFP-Halo and controls (His-GFP, bBSA) were spotted and incubated o/n

spotting pattern:

• spots were blocked 2x with 10 mg/mL BSA for 5 min
• sildes were blocked with 10 mg/mL BSA for 30 min
• slides were washed with PBS for 10 min
• slides were washed with 20 mM Imidazole in diluted PBS for 10 min
• slides were washed with diluted PBS for 10 min

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 Ni-NTA slides for measurement of cell free expressed proteins and purified antigens in iRIf

• prepared 2 PDITC slides according to protocol
• plasma activation: 80 L/h
• APTES incubation: 30 min
• PDITC incubation: 2 h
• slides were incubated with NTA-solution o/n

• blocking in APTES blocking solution for 1h at RT
• NiSO₄ incubation time: 1 h at RT
• slides were washed with PBS/diluted PBS

• used slide 424 and 029
• 3 µL of cell free expressed His-tYFP-Halo and controls (His-GFP, bBSA) were spotted and incubated o/n

spotting pattern:

• spots were blocked 2x with 10 mg/mL BSA for 5 min
• slides were blocked with 10 mg/mL BSA for 30 min
• slides were washed with PBS, imidazole in diluted PBS and in diluted PBS

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 Ni-NTA slides for measurement of cell free expressed proteins
• slide 024 and 216

• prepared 2 PDITC slides according to protocol
• plasma activation: 80 L/h
• APTES incubation: 30 min
• PDITC incubation: 2 h
• slides were incubated with NTA-solution o/n

• blocking in APTES blocking solution for 1h at RT
• NiSO₄ incubation time: 1 h at RT
• slides were washed with PBS/diluted PBS
• 3 µL of cell free expressed His-GFP and controls (His-GFP, bBSA) were spotted and incubated o/n

spotting pattern:

• spots were blocked 2x 5 min with 10 mg/mL BSA for 5 min
• slides were blocked 10 mg/mL BSA for 30 min

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 PDITC slides for measurement of purified antigens and PhyA in iRIf and bBSA in own setup

• prepared 2 PDITC slides according to protocol
• plasma activation: 80 L/h
• APTES incubation: 30 min
• PDITC incubation: 2 h
• slides were stored in desiccator o/n

• spotted the slides with 200 µg/mL bBSA
• 9 spots with bBSA, 1 spot with BSA (10 mg/mL) in the middle of the slide
• slides were incubated at 4 °C o/n

• blocked spots 2x with 10 mg/mL BSA for 20 min
• blocked slides with 10 mg/mL BSA for 45 min
• dried slides with wafergun and stored them under N₂-atmosphere till measurement

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 PDITC slides for measurement of purified antigens and PhyA in iRIf
• slide 265 and 426

• prepared 2 PDITC slides according to protocol
• plasma activation: 80 L/h
• APTES incubation: 30 min
• PDITC incubation: 2 h
• slides were stored in desiccator o/n

• antigens were diluted to a concentration of ~0.4-0.5 mg/mL
• 3µL of antigens and controls (GFP, bBSA) were spotted and icubated o/n
• spinned antigens before spotting

Spotting pattern:

Slide 265 - measured in new setup

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 PDITC slides for measurement of purified antigens and PhyA in iRIf and bBSA in own setup

• prepared 2 PDITC slides according to protocol
• plasma activation: 80 L/h
• APTES incubation: 30 min
• PDITC incubation: 2 h
• slides were stored in desiccator o/n

• spotted the slides with 200 µg/mL bBSA
• 9 spots with bBSA, 1 spot with BSA (10 mg/mL) in the middle of the slide
• slides were incubated at 4 °C for 11 h
• slides were blocked and afterwards measured in own setup

• protocols used :Plasma activation, iRIf slide preparation

• prepare 2 iRIf slides from Normann (309, 310)

• 6 iRIf slides were cleaned according to iRIf slide wash steps
• plasma activation for 5 min at 80 L/h gasflow
• incubated in APTES solution for 30 min
• incubation in PDITC solution for 2.5 h
• sandwiched slides with 80 µL NTA and incubated them at 4°C o/n

• blocked slides for 1 h in APTES blocking solution
• washed 2x in PBS for 10 min
• incubated in NiSO₄-solution for 1 h
• washed 1x in PBS for 10 min
• washed 2x in diluted PBS for 10 min then dried with wafer gun
• stored slides in slideholder under N₂-atmosphere for Norman (uses them the next day)

• protocols used :iRIf slide wash steps

• wash six of Jürgens iRIf slides (254, 466, 423, 426, 253 ,424) for first test with Halo

• 2 slides: 423, 466
• slides were cleaned with acetone, isopropanol and aqua dest
• plasma activation for 10 min at gas flow 60 L/h with valve 2 (air)
• slides were cross-sandwiched (Piehler style) with 15 µL of PLL-PEG-HTL for 15 min
• 3 µL of Halo-GFP-His, Halo-mCherry-His, bBSA, and 2 µL of pos./neg. controls (from Piehler: His-Halo-GFP/His-GFP) were spotted