Magnetic Bead Separations in Larger Volume Microplates

Magnetic Poles: Alternating or Not Alternating, That Is The Question

At V&P Scientific, we carry a series of Magnetic Bead Separation Blocks with a custom-designed bar magnet for separating larger volumes such as those in deep well microplates (DWPs). In this “M” series of bar magnet blocks, we have many to choose from where the number of bar magnets is different depending on the number of wells in the microplate and if the pellet needs to be divided into the two sides of the well or remain on only one side. The focus of this blog will be the 7-bar Magnetic Bead Separation Block shown below in figure 1 and its use with the larger volumes in DWPs.

“M” Series Magnetic Bead Separation Blocks

Part NumberNumber of MagnetsDeep Well MicroplateMag Bead Pellet Position
VP 771MAZM-14 bars6, 24, 48One side of the well
VP 771MCWZM-16 bars96One side of the well
VP 771MWZM-17 bars24, 48, 96Two opposing sides of the well (24, 48)
One side of the well (96)
VP 771MDWZM-113 bars96Two opposing sides of the well
Figure 2.

North Pole Up: All of these “M” series Magnetic Bead Separation Blocks are assembled with all of the bar magnets positioned north pole up. As we often do, we will create or modify one of our research tools based on a customer request (so don’t be shy about talking to us). In the case of the 7-bar magnet block VP 771MWZM-1 and a 48-deep well microplate with rectangular wells, they asked if we had tried to alternate the poles: north, south, north, south, etc.

Alternating North and South Poles: We found that by alternating the poles of the magnets in the 7 bar magnetic bead separation block (VP 771MWZM-1ALT), the separation of beads in a 48 well deep well plate with rectangular wells (10 ml per well) was significantly improved. This was characterized by keeping the beads off of the bottom of the well and more tightly collected next to the well’s vertical wall (see figure 4).

24 and 48 Well Deep Well Microplates: Recently a customer asked about a 24 well magnetic bead separation block. She also was using 48 well DWPs. Remembering a past customer who ordered a custom version of the 7 bar magnetic bead separation block VP 771MWZM-1ALT with the alternating magnetic poles had also used a 48 well DWP, I thought that maybe the 24 well DWP would work as well. A test with Beckman’s Agencourt GenFind magnetic beads and two different 24 well DWPs, one with round bottom wells (figure 5) and the other pyramid ones (figure 6), also resulted in improved separation with VP 771MWZM-1ALT. Beads that were encroaching on the lowest point of the well with the “all north up” magnets, were pulled up to the sides of the wells where the wall goes vertical when separated with the “north/south alternating” magnets. The proof is in the pictures!

96 Well Deep Well Microplates: Why stop with 24 and 48 well DWPs? What about 96 Well Deep Well Plates? A similar difference is seen for separation in a 96 well plate (figure 7 below), where having the magnetic poles alternating creates a a tighter collection of beads to the side.

Alternating Magnetic Poles are The Answer.
At least it is the answer for large volumes of magnetic beads in deep well microplates when using V&P’s 7-bar magnet block! So when you are doing magnetic bead assays, be they protein or nucleic acid isolations, and you have a large volume of sample in a twenty-four, forty-eight, or ninety-six well plate, the VP 771MWM-1ALT is the magnetic separation block to use.

And The Rest Of The Story:
To visualize the magnetic field lines we have a single well plate filled with iron filings that we place on top of the magnet separation block we want to look at. The iron filings will align with the magnetic lines of flux as they go from north pole to south pole.

In figure 8 you can see two bar magnets (of the 7-bar separation block VP 771MWZM-1) with north poles up in the photo on the left. The filings are all “lined up” along each bar magnet’s north pole edge. That is because the magnetic lines of flux are vertical as they extend from the poles. Since the south poles are on the opposite side of the bar magnet, the magnetic lines of flux have to reach up high and then bend around to the opposite ends of the entire separation block to find the south poles. This is in contrast to the photo on the right in figure 8 which shows the filings connecting the alternating pole magnets of a 7-bar separation block VP 771MWM-1ALT. Here you can see the filings are following the magnetic lines of flux as they reach toward each other over the gap between the magnets. This happens because the north and south poles are closer to each other and the magnetic lines of flux can just go right over! How does this relate to the magnetic bead separation tests I showed above? Imagine a 24 well positioned in that gap between the two bar magnets. If the iron filings are visualizing the magnetic “power” that is supposed to pull the beads over to the bars at the sides of the wells, then it is easy to see which magnetic pole configuration would work best!

Hydrophobic Coated vs Non-Coated Pin Transfers

Solid Pin Delivery Data For Aqueous Solutions In 96 Format With Uncoated And /Ah Hydrophobic Coated Pins
PinDescriptionnl TransferredCV%
0.229 mm diameter (FP9)Total PinUncoated7.412.4
Hydrophobic7.465.4
0.229 mm diameter (FP9)Hanging DropUncoatedN/AN/A
Hydrophobic2.093.8
0.457 mm diameter (FP1)Total PinUncoated33.483.2
Hydrophobic28.177.5
0.457 mm diameter (FP1)Hanging DropUncoated16.964.5
Hydrophobic8.510.8
0.787 mm diameter (FP3)Total PinUncoated87.323.9
Hydrophobic77.43.9
0.787 mm diameter (FP3)Hanging DropUncoated48.771.2
Hydrophobic43.059.4
1.19 mm diameter  (VP 409 & VP 386)Total PinUncoated247.222.8
Hydrophobic192.672.6
1.19 mm diameter (VP 409 & VP 386)Hanging DropUncoated76.351.6
Hydrophobic108.42.8
1.58 mm diameter (VP 408 & VP 384)Total PinUncoated273.54.6
Hydrophobic259.253.1
1.58 mm diameter (VP 408 & VP 384)Hanging DropUncoated201.935
Hydrophobic170.047.5

Transfer Of Horseradish Peroxidase In Tris Buffered Saline With Pin Tools

Conclusion

Coating pins will reduce the total amount of liquid transferred and also reduce the amount of non-specific binding to the stainless-steel pins. If the substance you are transferring has high non-specific binding this will be an important factor in selecting your pins.

Slot Pin Delivery Data For Aqueous Solutions In 96 Format With Uncoated And /Ah Hydrophobic Coated Pin
PinDescriptionnl TransferredCV%
0.229 mm diameter (FP9)Total PinUncoated7.412.4
Hydrophobic7.465.4
0.229 mm diameter (FP9)Hanging DropUncoatedN/AN/A
Hydrophobic2.093.8
0.457 mm diameter (FP1)Total PinUncoated33.483.2
Hydrophobic28.177.5
0.457 mm diameter (FP1)Hanging DropUncoated16.964.5
Hydrophobic8.510.8
0.787 mm diameter (FP3)Total PinUncoated87.323.9
Hydrophobic77.43.9
0.787 mm diameter (FP3)Hanging DropUncoated48.771.2
Hydrophobic43.059.4
1.19 mm diameter  (VP 409 & VP 386)Total PinUncoated247.222.8
Hydrophobic192.672.6
1.19 mm diameter (VP 409 & VP 386)Hanging DropUncoated76.351.6
Hydrophobic108.42.8
1.58 mm diameter (VP 408 & VP 384)Total PinUncoated273.54.6
Hydrophobic259.253.1
1.58 mm diameter (VP 408 & VP 384)Hanging DropUncoated201.935
Hydrophobic170.047.5

Transfer Of Horseradish Peroxidase In Tris Buffered Saline With Pin Tools

Conclusion

Although the slots in the pin are a precise volume, the liquid that is transferred is usually more. The reason for this is due to the surface tension of the liquid causing the liquid in the slot to “bow out” thus increasing the volume of the liquid in the slot. If is important for you to transfer exactly a certain volume we can make custom slots to match the surface tension characteristics of your liquid

Liquid Surface Tension

Effect Of DNA Or BSA Concentration On Slot Pin Transfers Of Uncoated And Hydrophobic Coated Pins (FP3CS500)
Solvent/SampleConcentrationCV%nl FITC TransferredCV%nl FITC Transferred
UncoatedUncoatedHydrophobic CoatedHydrophobic Coated
DMSO (-)08.1353.427.5298.72
DMSO + DNA (mg/ml)0.56.6497.216.6435.86
0.259432.494.1391.93
0.1258.9363.640.9344.75
0.06252.3381.862331.68
0.03131.5378.034.4331.71
0.01561.2357.521.4329.03
Tris (-)04.9577.317.2493.53
Tris + DNA (mg/ml)0.54.5540.531.1477.5
0.254.6518.216.1456.75
0.12515.8583.254.1438.82
0.06254.2551.173.1433.69
0.03134.4536.662.3458.37
0.01562.9528.531.2441.1
Tris + BSA (%)45.4462.1311409.27
14452.862.7426.58
0.2511.7456.451.3408.72
0.06251.1445.226.5393.07
0.01563.7462.853.9430.2
0.00391.5493.542.2437.29
0.0012.9504.250.7475.96
Conclusions

1. Increasing the concentration of DNA (sheared salmon sperm) to .25 mg/ml significantly increases the volume of DMSO liquid transferred for both coated and uncoated FP3S500 Slot Pins.
2. Increasing the concentration of DNA does not significantly increase the volume of Tris buffer (aqueous) transferred by both coated and uncoated FP3S500 Slot Pins.
3. Increasing the concentration of BSA (Bovine Serum Albumin) significantly decreases the volume of Tris buffer transferred by both coated and uncoated FP3S500 Slot Pins.
4. Hydrophobic coated FP3S500 Slot Pins transferred less DMSO – DNA and less Tris DNA and less Tris BSA than the uncoated FP3S500 Slot Pins.
5. Both coated and uncoated FP3S500 pins transfer significantly more aqueous solution than DMSO.

Effect Of DNA Or BSA Concentration On Slot Pin Transfers Of Uncoated And Hydrophobic Coated Pins (FP1CS50)
Solvent/SampleConcentrationCV%nl FITC TransferredCV%nl FITC Transferred
UncoatedUncoatedHydrophobic CoatedHydrophobic Coated
DMSO (-)04.249.382.149.31
DMSO + DNA (mg/ml)0.54.951.242.656.79
0.251.750.21.249.53
0.1251.551.272.349.77
0.06252.249.344.148.19
0.03131.249.030.250.23
0.01562.445.91.446.64
Tris (-)02.689.512.991.34
Tris + DNA (mg/ml)0.5777.110.684.62
0.253.982.221.684.89
0.1253.985.42185.08
0.06251.585.362.885.03
0.0313284.52388.19
0.01562.682.922.883.2
Conclusions

1. In contrast to the FP3S500 data, increasing the concentration of DNA to .25 mg/ml does not significantly increase the volume of DMSO liquid transferred for both coated and uncoated FP1S50 Slot Pins.
2. Increasing the concentration of DNA does not significantly increase the volume of Tris buffer (aqueous) transferred by both coated and uncoated FP1S50 Slot Pins.
3. In contrast to the FP3S500 data, FP1S50 coated pins transferred about the same volume of DNA at all concentrations as did uncoated pins.
4. Both coated and uncoated FP1S50 pins transfer significantly more aqueous solution than DMSO.
5. The differences between the FP3S500 and the FP1S50 pin may be due to the different pin diameter’s effect on contact angle and therefore on the “wetting” of the pin. See the diagram on the link to / ah energy system.

PinDescriptionnl TransferredCV%
0.229 mm diameter (FP9)Total PinUncoated7.412.4
Hydrophobic7.465.4
0.229 mm diameter (FP9)Hanging DropUncoatedN/AN/A
Hydrophobic2.093.8
0.457 mm diameter (FP1)Total PinUncoated33.483.2
Hydrophobic28.177.5
0.457 mm diameter (FP1)Hanging DropUncoated16.964.5
Hydrophobic8.510.8
0.787 mm diameter (FP3)Total PinUncoated87.323.9
Hydrophobic77.43.9
0.787 mm diameter (FP3)Hanging DropUncoated48.771.2
Hydrophobic43.059.4
1.19 mm diameter  (VP 409 & VP 386)Total PinUncoated247.222.8
Hydrophobic192.672.6
1.19 mm diameter (VP 409 & VP 386)Hanging DropUncoated76.351.6
Hydrophobic108.42.8
1.58 mm diameter (VP 408 & VP 384)Total PinUncoated273.54.6
Hydrophobic259.253.1
1.58 mm diameter (VP 408 & VP 384)Hanging DropUncoated201.935
Hydrophobic170.047.5

Aqueous Transfer with Solid Pins

Hydrophobic coating pins will reduce the total amount of aqueous HRP liquid transferred and also reduce the amount of non-specific binding to the stainless-steel pins. If the substance you are transferring has high non-specific binding this will be an important factor in selecting your pins.

 

Pin diameter also has an effect on the degree of reduction of liquid transfer with hydrophobic coating as the smaller the diameter the less the reduction of transfer. This is most likely due to the curvature of the pin affecting the wetting contact angle

PinDescriptionnl TransferredCV%
0.457 mm diameter (FP1)6 nl SlotTotal Pin*Uncoated25.610.8
HydrophobicN/AN/A
10 nl SlotTotal Pin*Uncoated23.366.1
Hydrophobic25.856.9
50 nl SlotTotal Pin*Uncoated67.832.5
HydrophobicN/AN/A
0.787 mm diameter (FP3)  100 nl SlotTotal Pin*Uncoated180.327.2
Hydrophobic205.845.5
200 nl SlotTotal Pin*Uncoated277.824.9
Hydrophobic287.33.8
500 nl SlotTotal Pin*Uncoated581.165.2
Hydrophobic555.693

DMSO Transfer with Slot Pins

Hydrophobic coating pins will slightly increase the total amount of DMSO FITC liquid transferred.

PinDescriptionnl TransferredCV%
0.787 mm diameter (FP3)    100 nl Slot Total Pin, Including SlotUncoated195.691.6
Hydrophobic170.22.9
0.787 mm diameter (FP3)  100 nl Slot, Slot OnlyUncoated149.674.9
Hydrophobic129.617.6
0.787 mm diameter (FP3)200 nl Slot Total Pin, Including SlotUncoated269.771.9
Hydrophobic228.6217.1
0.787 mm diameter (FP3)200 nl Slot, Slot OnlyUncoated237.528.9
Hydrophobic186.95.9

Aqueous Transfer with Slot Pins

Although the slots in the pin are a precise volume, the liquid that is transferred is usually more because of the volume carried on the sides of the pins. 

As seen with other aqueous data the amount transferred on hydrophobic coated Slot pins is less than on uncoated Solid or Slot pins. Thus Hydrophobic coating has the most effect on aqueous transfers.

Withdrawl Speeds Impact on Volume Transfer

Solid Pins More affected by Source Plate Volume

Volume Transferred For FP1 Pins (Uncoated) In 96 And 384 Formats
Volume Transferred For FP3 Pins (Uncoated) In 96 And 384 Formats

Note: Same volume (200ul for 96 Format and 74 ul for 384 Format) in recipient plates and same pin withdrawal speed for all pins. Changes to pin withdrawal speed or volume in the source plate can result in different volumes being transferred.

Transfer volumes should always be confirmed by customers for their assay conditions and automated system.

Aqueous Solutions Pin Transfer Volumes Ranges

Aqueous Solutions on Uncoated Pins in 96 Format Microplates(1)
Pin TypePin Diameter(mm)Shape96 Format Low Range(nL)²96 Format High Range(nL)²
FP90.229Solid1339
FP80.356Solid1537
FP10.457Solid2261
FP1S60.4576nL Slot3467
FP1S100.45710nL Slot3974
FP1S500.45750nL Slot90124
FP30.787Solid93213
FP3S1000.787100nL Slot213334
FP3S2000.787200nL Slot311449
FP3S5000.787500nL Slot515671
FP40.914Solid126289
Footnotes: (1) Delivery volume range is determined by speed of withdrawal from source liquid: Z-Speed Range = 1.5-30 mm/sec, slow speed = low volume delivery range, fast speed = high volume delivery range (2) 200ul source plate volume per well
Aqueous Solutions on Hydrophobic Pins in 96 Format Microplates(1)
Pin TypePin Diameter(mm)Shape96 Format Low Range(nL)²96 Format High Range(nL)²
FP90.229Solid1338
FP80.356Solid
FP10.457Solid2360
FP1S60.4576nL Slot3367
FP1S100.45710nL Slot4075
FP1S500.45750nL Slot86119
FP30.787Solid76209
FP3S1000.787100nL Slot188324
FP3S2000.787200nL Slot288436
FP3S5000.787500nL Slot473649
FP40.914Solid
Footnotes: (1) Delivery volume range is determined by speed of withdrawal from source liquid: Z-Speed Range = 1.5-30 mm/sec, slow speed = low volume delivery range, fast speed = high volume delivery range (2) 200ul source plate volume per well
Aqueous Solution on E-Clip, Uncoated Pins(1)
Pin TypePin Diameter(mm)ShapeLow Range(nL)²High Range(nL)²
FP1.58Solid Pointed175594
FPS.51.58500nL Slot524962
FPS1.581000nL Slot10561476
FPS21.582000nL Slot17392174
FPS51.585000nL Slot51504953
FP61.58Solid Flat465960
FP6S.51.58500nL Slot9341445
FP6S1.581000nL Slot13961930
FP6S21.582000nL Slot20722637
FP6S51.585000nL Slot48204693
Footnotes:(1) Delivery volume range is determined by speed of withdrawal from source liquid: Z-Speed Range = 1.5-30 mm/sec, slow speed = low volume delivery range, fast speed = high volume delivery range (2) 200ul source plate volume per well for 96 Format and 75ul source plate volume per well for 384 Format

DMSO Pin Transfer Volume Range Charts

Uncoated Pins in 96 and 384 Format Microplates(1)
Pin TypePin Diameter(mm)Shape96 Format Low Range(nL)²96 Format High Range(nL)²384 Format Low Range(nL)³384 Format High Range(nL)³
FP90.229Solid41038
FP80.35Solid1326618
FP10.457Solid18431131
FP1S60.4576nL Slot24491534
FP1S100.45710nL Slot30542140
FP1S200.45720nL Slot37612746
FP1S300.45730nL Slot46683554
FP1S400.45740nL Slot57784563
FP1S500.45750nL Slot70905675
FP30.787Solid671392979
FP40.91Solid941973498
FP3S1000.787100nL Slot175241114163
FP3S2000.787200nL Slot280332203250
FP3S5000.787500nL Slot535559427464
FP4S10000.911000nL Slot9401011704800
FP4S20000.912000nL Slot1518160812771362
Footnotes: (1) Delivery volume range is determined by speed of withdrawal from source liquid: Z-Speed Range = 1.5-30 mm/sec, slow speed = low volume delivery range, fast speed = high volume delivery range (2) 200ul source plate volume per well (3) 75ul source plate volume per well
Hydrophobic-coated Pins in 96 and 384 Format Microplates(1)
Pin TypePin Diameter (mm)Shape96 Format Low Range(nL)²96 Format High Range(nL)²384 Format Low Range(nL)³384 Format High Range(nL)³
FP9H0.229Solid41038
FP8H0.35Solid924617
FP1H0.457Solid1539927
FP1S6H0.4576nL Slot23491432
FP1S10H0.45710nL Slot29532038
FP1S20H0.45720nL Slot35592643
FP1S30H0.45730nL Slot47693553
FP1S40H0.45740nL Slot54754158
FP1S50H0.45750nL Slot69905773
FP3H0.787Solid671342776
FP4H0.91Solid9518932102
FP3S100H0.787100nL Slot170227108164
FP3S200H0.787200nL Slot266320190239
FP3S500H0.787500nL Slot520542416456
FP4S1000H0.911000nL Slot9321000741805
FP4S2000H0.912000nL Slot1571163813511423
Footnotes: (1) Delivery volume range is determined by speed of withdrawal from source liquid: Z-Speed Range = 1.5-30 mm/sec, slow speed = low volume delivery range, fast speed = high volume delivery range (2) 200ul source plate volume per well (3) 75ul source plate volume per well
E-Clip, Uncoated Pins, for 96 and 384 Format Microplates(1)
Pin TypeDiameter (mm)Shape96 Format Low Range(nL)²96 Format High Range(nL)²384 Format Low Range(nL)³384 Format High Range(nL)³
FP1.58Solid Pointed147411168395
FPS.51.58500nL Slot442704631843
FPS1.581000nL Slot893113013431498
FPS21.582000nL Slot1911203826072767
FPS51.585000nL Slot3908429651805253
FP61.58Solid Flat323674154398
FP6S.51.58500nL Slot73410428551053
FP6S1.581000nL Slot1210150016381717
FP6S21.582000nL Slot2299238427873068
FP6S51.585000nL Slot4329465652375245
Footnotes:(1) Delivery volume range is determined by speed of withdrawal from source liquid: Z-Speed Range = 1.5-30 mm/sec, slow speed = low volume delivery range, fast speed = high volume delivery range (2) 200ul source plate volume per well (3) 75ul source plate volume per well