Applications - Presentations

MicroFill: A 96-/384-Well Microplate Reagent Dispenser for HTS and Drug Discovery

06-Apr-04

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Related Products: MicroFill

 

This poster shown at Lab Automation, January, 2002

 

The MicroFill is a reagent dispenser capable of dispensing to either 96- or 384-well microplates without any hardware changes and 24-well plates with a manifold change. Additionally, the MicroFill is capable of dispensing to deep-well microplates by changing the plate carrier. In this monograph, we will discuss the MicroFill’s speed, accuracy, and precision at fluid volumes that span its reported range. The MicroFill is provided as two different models (standard and autoclavable) to meet the diverse needs of today’s investigator. Besides the standard model, BioTek has developed the autoclavable model in response to the need for easy sterilization of the MicroFill’s fluid path. The syringe barrel and piston have been located externally, and along with the tubing, check valves and manifold have been designed to be fully compatible to autoclaving. This allows the user to steam-sterilize the entire fluid path instead of using chemical sterilization methods prior to dispensing sterile solutions. Additionally, the autoclavable version of the MicroFill offers increased organic resistance. Using the newly designed manifold and syringe pump in conjunction with optional chemical-resistant check valves allows the MicroFill to be used with agents such as dimethyl sulfoxide (DMSO) and acetonitrile.

 

Introduction

 

Today’s biomedical research requires instrumentation that is both functional and versatile. While high throughput screening (HTS) and drug discovery laboratories require instrumentation that can be automated, pilot assay laboratories may not necessarily need total automation. Towards that end, BioTek has developed the MicroFill, (pronounced “micro” Fill) reagent dispenser capable of running stand-alone or computer controlled as part of a robotics system (Figure 1). The MicroFill is compatible with both conventional 96- and 384-well microplates, and by removing the standard plate holder, can also dispense into deep-well microplates. The use of a specialized 8-channel manifold also allows the MicroFill to be used with half-area 96-well plates and 24-well plates. The microprocessor - controlled syringe pump is based on a tested, low maintenance design that requires no recalibration, yet provides a high degree of accuracy and precision. As part of the manufacturing process, BioTek performs a 6-point calibration procedure to accurately tune the syringe pump over a wide range of volumes. Among the many variables which programming allows, is the control of flow rates from (225 Microl/well/sec), for dispensing to cell cultures, to (1000 Microl/well/sec) for rapid and vigorous reagent dispensing. The flexible software provides complete programming capabilities from the keypad. For more complete automation, robotic interfaces can be developed using ActiveX® software commands. The μFill’s diminutive size, with a 14 x 14-inch footprint and a height of 7 inches, allows it to be used almost anywhere.

 

Figure 1. MicroFill 96-/384-Well Microplate Dispenser.

 

Materials and Methods

 

Dispense accuracy and precision was determined using a combination of a gravimetric method and the absorbance of dye solutions. Gravimetric determinations were performed by weighing plates using a Sartorius A 120S analytical balance, while the absorbance of a dye solution (FD&C Blue No. 1) was used to estimate the dispense precision of the MicroFill Microplate Dispenser. The dispense-volume accuracy into 96- or 384-well microplates was determined by weighing an empty plate before and the same plate after the dispense cycle by the MicroFill. The average weight per well was calculated by dividing difference between initial and final weights (Delta) by the total number of wells (96 or 384). Using the specific gravity of water (1 g/ml) a conversion from weight to volume was then made. Next, deionized water was added to each well of the microplate such that the final volume was expected to be 300 Microl or 100 Microl for 96-well and 384-well plates respectively. Note that the volume added varied depending on the intended dispense-volume programmed. The absorbance at 630 nm (450 nm reference) of all the wells in the 96- or 384-well microplate was measured using a Synergy HT Multidetection Reader (BioTek Instruments) and the average calculated. A plate-specific factor was then calculated by dividing the average per-well absorbance by the per-well dispense volume. This factor was then used as a conversion factor to calculate the dispense volume of each well from its absorbance.

Tests involving the use of Sephadex beads were done as follows. Briefly, a mixture of deionized water and hydrated Sephadex G-50 Superfine beads (Amersham Pharmacia Biotech, Piscataway, New Jersey) was stirred using a magnetic stirrer at a setting sufficient to prevent the slurry from settling. The aspiration tubing was situated such that the end was located approximately 2.5 cm (1 inch) from the bottom of the reservoir. It was found that maintaining the slurry in an agitated state was paramount to accurate dispensing of the beads. If there was a delay of greater than 10 minutes between plates, the MicroFill was re-primed using the “New Buffer Prime” software selection. Note that beads lost through priming can be saved using the drain hole and tubing to a collection container. All gravimetric experiments were performed as described previously. It was also observed that light scattering by the beads could be used to measure the volume of bead dispense optically. While several different wavelengths were tested, 405 nm appeared to provide the most reliable results. Using a MicroQuant microplate spectrophotometer (BioTek Instruments, Winooski, VT), the absorbance of samples at 405 nm were measured and the results compared to a previously prepared standard curve and the dispense volume was interpolated.

 

Results

 

The time necessary for routine dispensing by the MicroFill was examined for various volumes at both the slowest and fastest dispense rates. The MicroFill can dispense 10 Microl into each well of a 96-well microplate in less than 8 seconds and 5 Microl into a 384-well microplate in approximately 10.5 seconds. This time includes the time required to move the plate carrier from the home position to the manifold at the beginning of the dispense cycle, as well as returning to the home position at the completion of the cycle. As one might imagine, with larger per-well volumes the time required to fill a plate increases. Dispense-times for deep-well plates were also measured at volumes of 500, 1000, and 2000 Microl per well. As demonstrated in Table 1, the time necessary for 300 Microl to be dispensed into all 96 wells is 44.5 seconds at rate 1. However, at larger per-well volumes faster fluid rates are available which can cut the dispense time in as much as half. The degree of increase is dependent on the volume being dispensed, with larger volumes showing the greatest increase. Similar results were seen when 384-well plates were used. The standard model of the MicroFill Reagent Dispenser has five different dispense rates that affect the time required to fill a plate. The autoclavable model of the MicroFill uses a different syringe design that allows for faster dispense speeds. At the lowest volumes, the autoclavable MicroFill can dispense 10 Microl into each well of a 96-well microplate approximately 0.5 seconds faster than the standard model (7.1 vs. 7.6 seconds) and for a 5 Microl dispense into a 384-well microplate, it is approximately 0.7 seconds faster (Table 1). However, at larger volumes, the time savings from the autoclavable model become quite dramatic. For example, when dispensing 1000 Microl into a deep-well 96-well microplate, the autoclavable model requires approximately 50 seconds to complete the task, while the standard model needs 135 seconds.

96-Well Dispense Times#

348-Well Dispense Times#

Microl Standard Autoclavable Microl Standard Autoclavable
Rate (Microl/well/sec) Rate (Microl/well/sec) Rate (Microl/well/sec) Rate (Microl/well/sec)
450 1000 450 1000 225 500 225 500
10 7.6 --- 7.1 --- 5 10.4 --- 9.7 ---
20 8.1 --- 7.3 --- 10 12.7 --- 11.4 ---
30 9.8 --- 8.2 --- 20 17.9 --- 16.4 ---
40 11.0 --- 9.7 --- 30 22.8 --- 18.9 ---
50 12.1 --- 10.9 --- 40 28.1 --- 18.7 17.0
100 18/7 --- 11.7 10.7 50 33.6 --- 20.9 18.8
200 31.7 17.8 17.3 14.9 60 38.7 24.0 23.1 20.2
300 44.5 22.8 20.0 16.0 70 44.0 26.1 25.4 22.0
500 70.8 33.9 28.7 21.6 80 49.2 28.2 27.6 23.6
1000 135.6 59.6 50.1 35.6 90 54.3 30.1 29.8 25.3
2000 --- 115.4 97.5 68.8 100 59.5 31.9 32.0 27.0

# Note that all times are in seconds.

 

Table 1. Dispense times of Standard and Autoclavable models of the MicroFill.

The accuracy and precision of the MicroFill were determined using a number of different techniques. As demonstrated in Table 2, the MicroFill is accurate across the entire range of its volume settings for 96-well plates. When the minimum setting for 96-well plates (10 Microl) was selected, the dispense-volume, determined gravimetrically, was found to deviate from expected by less than 3%. The deviation diminished with larger volumes to less than 1%. In all cases, the dispense volume was quite precise.

 

Dispense Accuracy of the MicroFill Dispenser into 96-Well Plates

Expected Volume (Microl) Calculated Volume (Microl)# % Deviation
10 10.3 + 0.34 2.97
20 19.9 + 0.57 0.34
30 29.4 + 0.71 2.03
40 40.2 + 1.09 0.50
50 49.9 + 1.17 0.12
60 59.9 + 1.23 0.20
70 69.9 + 1.61 0.17
80 80.2 + 0.91 0.27
90 90.1 + 1.92 0.01
100 100.1 + 2.03 0.12
150 150.3 + 2.97 0.18
200 199.8 + 3.92 0.12
250 250.3 + 3.74 0.11
300 300.4 + 3.17 0.14
500 497.9 + 0.22 0.40
1000 997.6 + 0.60 0.20

# Note that these data represent the mean and average of twelve determinations.

Table 2. Dispense accuracy into 96-Well plates.

Figure 2. Dispense precision into 96-Well plates using the MicroFill 96-/384-Well Reagent Dispenser at various dispense volumes.

 

As demonstrated in Figure 2, the coefficient of variance (%CV) was found to be less than 4% at 10 Microl per well. The %CV also decreased with larger volumes to less than 2% at volumes above 100 Microl per well. When the dispense accuracy into 384-well microplates was examined, similar results were found. When dispensing 5 Microl into each well of a 384-well plate, the average deviation was calculated to be approximately 1% (Table 3). Again, as the per-well volume was increased the deviation from expected diminished to less than 1%. The precision when dispensing into 384-well plates was also quite good, with CVs being less than 4% at the lowest volume setting (Figure 3). Because the MicroFill enables the selection of different fluid dispense-rates, the accuracy of pipetting different volumes at different rates was examined.

 

Dispense Accuracy of the MicroFill Dispenser into 384-Well Plates

Expected Volume (Microl) Calculated Volume (Microl)& % Deviation
5 4.95 + 0.30 1.01
10 9.76 + 0.22 2.43
20 20.22 + 0.62 1.12
40 29.87 + 0.79 0.45
50 39.96 + 0.69 0.11
60 50.07 + 0.86 0.13
70 60.00 + 0.95 0.01
80 79.92 + 1.05 0.11
90 89.84 + 0.85 0.18
100 99.88 + 1.18 0.12

& Note that these data represent the mean and average of 384 determinations.

 

Table 3. Dispense accuracy into 384-Well plates.

Figure 3. demonstrates the dispense precision into 384-well plates. The increase in absolute values most likely reflects the natural variation of the meniscus of the solution in microplate wells. Note that in order to obtain adequate coverage of the wells with solution, it was necessary to add deionized water to all wells such that the final volume was 100 Microl.

Figure 3. Dispense precision into 384-Well plates using the MicroFill 96-/384-Well Reagent Dispenser at various dispense volumes.

 

The autoclavable version of the MicroFill, besides providing an easy means for sterility, also provides an easy access for cleaning purposes. Removal of the syringe piston and barrel is easily accomplished. As demonstrated in Figure 4A, the external syringe is located at the rear of the MicroFill, protruding approximately 3 inches. The barrel is attached with two hex-screws that are removed with the provided hex-wrench (Figure 4B). Once the barrel has been removed, the piston is exposed and can be removed after loosening a setscrew, using the same wrench (Figure 4C). The barrel, piston, tubing, check-valves, and dispense manifold can then be sterilized by autoclaving. One can expect that these parts will provide accurate liquid-dispenses after a minimum of 50 autoclave-sterilization cycles. In addition, extra components of the fluid-path assembly can be purchased, allowing for sterilization of one set while another set is in use.

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 4. External syringe unit located at the rear of the autoclavable model of the MicroFill Reagent Dispenser.

Sephadex Bead Dispense Accuracy into 96-Well Plates

Gravimetric Method

Absorbance Method

Expected (Microl/well) Volume (Microl) % CV Expected (Microl/well) Volume (Microl) % CV
100 100.39 0.399 100 106.63 5.32
200 201.72 0.228 200 202.00 1.38

 

Table 4. Dispense accuracy of the MicroFill for Sephadex into 96-Well Microplates.

 

Figure 5. Microplate strip with Dispensed Sephadex beads.

 

Sephadex Bead Dispense Accuracy into 384-Well Plates

Gravimetric Method

Absorbance Method

Expected (Microl/well) Volume (Microl)

 



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