Rather than using physical wells, DropArray microplates are completely flat including a series of 96 or 384 hydrophilic spots. A hydrophobic surface occupies the entire flat surface area of the plate except for the spots. These spots form an array that conform to the well layout of the ANSI/SBS 4-2004 microplate standard. Liquid dispensed onto these spots form droplets that remain in a fixed position from the surface tension created from the hydrophobic surroundings, even when the plate is shaken or inverted. The DropArray microplate only has walls around the outside of the plate, which serve to contain wash buffers added in bulk during wash steps of the assay. DropArray offers three platforms that address unmet needs for cell processing with flow cytometry (DA-Cell), miniaturizes and improves Luminex and other bead-based assays (DA-Bead), and enables reduction of cells and expensive reagents for high content screening (DA-HCS).
DropArray microplates enable simple and easy miniaturization of assays at 2-4 microliters for the 384-well format and 5-15 microliters for the 96-well format because of their unique wall-less design. The wall-less feature eliminates capillary action, the biggest barrier to miniaturization with traditional microplates. Furthermore, the wall-less feature allows extremely gentle washing by liquid exchange, which is different from the harsh, convection-based washing performed by a microplate washer with an array of nozzles.
Unlike a standard microplate that requires dispensing and aspiration from each well, bulk liquids like wash buffers are added to or removed from the entire DropArray microplate at once. By reducing turbulence in wash steps, far fewer cells and beads are lost from the assay droplets, even with suspension cells and non-magnetic beads compared to traditional microplates. Evaporation is controlled using an Anti-Evaporation Lid which provides a humidified environment and minimizes evaporation of the droplets.
What pipette do you recommend to handle 5—15 microliters of sample and reagent for DropArray microplates?
Among all the pipettes available in the market, Curiox users find that Rainin® pipette is best at handling small volumes of 5 – 15 microliters, particularly when a solution is sticky or viscous due to the high content of proteins and additives. Rainin pipette tips have a long narrow body and small orifice which minimizes a chance of wicking of a solution onto a tip. Pipettes of other brands and suppliers can be used but they need a bit of skill to precisely handle small volumes.
A DropArray microplate is as easy to use as a traditional microplate because the hydrophilic spots on the plate attract reagents while the hydrophobic surface between the spots repels reagents. A dispensed droplet adheres to a spot and does not come off during the typical handling expected for a plate. When dispensing reagents at small volumes for bead-based assay applications or replacing centrifugation for flow cytometry cell processing, DropArray improves workflow over traditional methods.
A DropArray Washing Station typically needs ~110 ml for a 1x washing cycle. A 1x wash cycle and 3x wash cycles need ~2 min and ~4 min respectively, including 30 seconds of built-in rest time needed before starting the next wash process for precipitating beads onto the surface of a plate.
DA-Cell for Flow Cytometry
The minimum time for optimized cell recovery is recommended at 20 minutes. The addition of 10 to 20 minutes of incubation time reduces the loss of cells to less than 0.1% per wash step.
Yes, the DA-Cell plate is compatible with any cytometer that has a 96-well plate loader.
We are interested in rare cells and events and usually need larger volumes. Can we combine wells to get the number of rare cells we need for our studies?
Using traditional centrifuge methods, cell recovery is poor and rare cells can be lost. With the DA-Cell plate and washer, greater than 95% recovery of cells is achieved and hence the starting number of cells and volumes can be minimized. As a rule of thumb, a user needs about 50 % of cell numbers required for a conventional plate due to ~50 % cell loss by a conventional method. Furthermore, if a number of cells required is bigger than that of one well, then a user can pull the cells from multiple wells as needed in order to reach the desirable number of cells to process.
Yes, per your usual protocol the fixing, permeabilization, and staining can be performed all on the DA-Cell plate.
The plate can be run in full or partially. If only a few wells are used, the plate can be stored at 4°C and the remainder of the wells can be used later. However, wells cannot be reused and caution must be taken to document the wells that have been used. Once all the wells are used, they can’t be reused.
How do the cells stay in the well and not get washed away with the laminar flow created by the dual-nozzle action?
DropArray (or DA) plate has a unique patent-protected wall-less design. The absence or minimal presence of a wall allows for laminar flow during the addition and aspiration of a liquid. By nature of a laminar flow, the velocity of the flow reaches zero at the surface of a plate where cells are resting by gravity. Such low velocity allows cells at the surface to remain where they are even if the number of cells is as small as one. Yet, the bulk of existing liquid is replaced by an incoming liquid effectively rinsing the spot and cells.
The entire wash protocol has been optimized to combine the unique, patented features of wall-less plate as well as the flow rate and incubation time to obtain the greatest cell recovery. Changing the flowrate is possible via the firmware but is not accessible from the user interface and not recommended.
The DA-Cell well can hold up to 70 µl and up to approximately 5 million cells. During the protocol, the DA-Cell plate is vortexed and the working volume range above has been validated by our scientist. Volumes above 70 µl are not recommended and may jeopardize the ability of the spot to be retained on the wall-less plate during vortex of a plate.
DA-Bead for Luminex Assays
I am using CBA®, Luminex®, Singulex® or Quanterix® assays, which use beads for immunoassay. How would my assay benefit by using DA-Bead microplate?
DA-Bead is a unique washer and wall-less plate system that allows simple and easy miniaturization by simply reducing the volume of samples and reagents by 5 – 10 fold under the same workflow. The resulting data from miniaturized assays are comparable or improved from those on a traditional microplate depending on the type of an assay, samples and target analytes. For the details about Luminex and Singulex assay, please see a section dedicated for each application below. For other bead-based assays, please contact Curiox’s representative for the details.
It appears that handling 5 – 10 microliters of solutions by manual pipetting is not practical. Wouldn’t it require automated liquid handling instrument to dispense such small volumes, particularly with sticky biological samples?
Handling of 5 – 10 microliters of solutions with a traditional plate or tube is not easy as a traditional microplate is designed to hold a much larger volume. The DropArray DA-Bead microplate is designed to facilitate handling small volumes of solutions specifically. With the help of Rainin pipettes, DropArray users have found that the handling of small volumes is not different from larger volumes for a traditional plate.
Yes. The DropArray HT Washing Station (HT200) has a PC-HT200 RS232C port for communication with any automation controllers.
What are the benefits of using DropArray DA-Bead plates for running Luminex assays such as Milliplex®, Bio-Plex®, Procartaplex® assays?
A DA-Bead plate improves reproducibility, limit of detection, and reduces the consumption of both samples and beads to 5 microliters each. The improvement of reproducibility and limited of detection is particularly distinctive with samples of body fluids containing matrix. These benefits are a result of dispersion of beads on the surface of the drop to allow optimal reaction kinetics and incubation and better washing with no bead loss. Furthermore, the consumption of only 5 microliters of sample per plate droplet makes DA-Bead plates ideal for precious samples such as mouse sera and human tears.
Wouldn’t dispensing of 5 microliters of sample and bead reagent increase my CV compared to dispensing 25 microliters on a traditional microplate?
CVs are a result of variability and this variability can come from the user or the assay. In general, dispensing smaller volumes has more error than dispensing larger volumes, but this is very user/technique-dependent and pipette quality driven. It is known that the higher intra-assay CVs for Luminex assays stem from a combination of steps, mostly from dispensing, washing, and Ab-Ag binding on the bead surface. While the DropArray system may produce higher CVs purely due to dispensing in smaller volumes, it achieves comparable CVs overall thanks to uniform and consistent specific binding with minimal non-specific binding and better washing with no loss of beads. Overall, DropArray system achieves comparable, if not lower, CVs thanks to uniquely improved incubation and washing.
Why doesn’t miniaturization of a Luminex assay on DA-Bead plates affect the sensitivity? Wouldn’t the reduction of sample volume decrease the sensitivity as it has a smaller amount of a target analyte?
The miniaturization of a Luminex assay does not affect the sensitivity. In a Luminex assay, the ratio of sample volume to the number of beads is determined at 25 microliters of sample for ~2,500 beads. When an assay is miniaturized by 5-fold on a DA-Bead plate, both the sample and the number of beads are reduced correspondingly. It leaves the same amount of a target analyte available per bead. Because of a smaller volume helping to reach equilibrium fast, the superior washing, and minimizing background signal, the sensitivity is at least equivalent or, in some limited cases, improved compared to the traditional microplate method.
DA-Bead plate may show better sensitivity for certain analytes for certain types of samples while showing comparable sensitivity at a miminum. Generally, DA-Bead plate shows better reproducibility. The features that enable better sensitivity and/or reproducibility are 1) Incubation of a sample and beads at total 10-15 microliter volume with beads retained on a surface during shaking, and 2) Thorough washing of individual beads by laminar flow with no loss of beads and less residual fluid compared to traditional microplates.
Performance of a Luminex assay are affected by a number of factors including good lab practice. However, there are two inherent challenges that good lab practice may not be able to resolve. First, the mixing of beads can be inconsistent due to the various components or viscosities of a sample solution during the incubation span producing variability between samples. Because the DA-Bead plate enables homogeneous incubation with the beads retained on a surface, DropArray technology achieves superior reproducibility. For example, when a Luminex assay runs on a DA-Bead plate, beads never aggregate as they are retained on a surface instead of floating and coming together to aggregate, ensuring reproducibility regardless of sample types. Second, thorough washing of individual beads by a laminar flow ensures consistent and reproducible binding of a target analyte with minimal non-specific binding.
I can’t see how the incubation of beads retained on a surface produces comparable sensitivity than beads freely floating in a solution. How can it be possible?
The method of incubating beads retained on a surface by magnet was originally developed to improve the reproducibility of the assay regardless of sample types and eliminate bead aggregation. The small volume of a drop on DA-Bead plate allows thorough and uniform mixing of beads with analytes even when beads are retained on a surface. In addition, when beads are retained on a surface, they attract less non-specific binding, leading to better sensitivity relatively. This provides similar performance characteristics of planar array assays of enhanced sensitivity while allowing the use of the more flexible format, bead-based arrays.
If DA-Bead plate shows less matrix effect, then does it mean that my sub-optimized assay buffer that shows matrix effect may work well on a DA-Bead plate?
It is possible that a sub-optimal assay buffer may be good enough on a DA-Bead plate with minimal matrix effect. In a number of Luminex assays, a same matrix buffer which showed matrix effect on a traditional microplate showed minimal matrix effect when used with a DA-Bead plate.
If the magnetic retention of beads on a surface is partly responsible for the better data, can I use the method for my Luminex assay on a traditional microplate?
Based on preliminary data from Curiox’s team, this method does not work on a traditional 96-well microplate because there is another critical factor missing for a microplate – small volume and wall-less feature. The small 1/5th volume and wall-less feature on DA-Bead plate enable thorough mixing and binding even when beads are retained on a surface. On a microtiter plate, the large volume and presence of a wall and corners appear to hinder thorough mixing and binding when beads are retained on a surface.
I am using high-plex assay of 40- to 80-plex. Will the incubation and washing claimed by DA-Bead plate work well as beads may pile up on each other because the surface area is too small?
While the surface area of DA-Bead plate is smaller than that of microtiter plate, it still offers enough surface area. Several users of a DA-Bead plate have successfully run commercial and customized Luminex assays from 40-plex up to 300-plex achieving the same quality data as lower plex assays.
Are there enough beads when a Luminex assay is reduced to 5 microliter bead solution on a DA-Bead plate?
Yes. In a 25 microliter bead solution, there are ~2,500 beads per analyte. A 5 microliter bead solution contains ~500 beads per analyte, which is still excessive for the number of beads required for reading, which is generally between 50 and 100.
Would reading of a DA-Bead plate by a Luminex reader take longer than a microtiter plate because a DA-Bead plate has only 1/5th beads per analyte per well?
The reading time of a DA-Bead plate is not affected by the reduced number of beads in general thanks to the optimization of reading parameters and less bead aggregation compared to a traditional microplate. However, Curiox’s team has found that some of MagPix® readers may take 20 % longer time to read due to the constraints of its firmware.
Does the whole-plate washing of a DropArray DA-Bead plate cause cross-contamination between droplets?
No. A number of tests performed by Curiox’s R&D team and DropArray users have demonstrated no detectable cross-contamination between droplets. In the perspective of cross-contamination, there are two possible pathways: cross-contamination of beads or samples. The cross-contamination of beads has been tested 1) by dispensing an excessive number of beads in alternate drop on a plate with blank drops on the rest of the plate and 2) by dispensing ~10 beads per drop onto an entire plate and physically count. In both cases, beads were observed to stay where they are after 10x wash cycles, suggesting that there is no bead loss nor cross-contamination between drops. The cross-contamination of samples has been tested 1) by dispensing HRP enzyme on column 2 and blank buffer on the rest of a plate followed by the addition of a chemifluorescent substrate and 2) by running a Luminex assay with an analyte of an extremely high concentration on one spot and a blank buffer on the rest of a plate. In both cases, the blank spots right next to the control spots showed same background signals as those farthest from control spots, suggesting no detectable cross-contamination of reagents to neighboring spots.
No. The entire workflow of a Luminex assay on a DA-Bead plate is identical to that of a conventional microtiter plate. The DA plate replaces the traditional microplate and the DropArray Washing Station is used during the washing steps. You can watch a video of the entire assay run on a DA-Bead plate at www.curiox.com/videos.html
DA-Bead for Singulex Assays
A DA-Bead plate eases the workflow of Singulex assay and reduces the needs of samples by 5 – 10 fold to 10 – 20 microliters and beads by 10 fold to 10 microliters. In the workflow, the maximum wash time is reduced to ~6 minutes instead of 20 minutes on a traditional plate thanks to effective and efficient wash by DropArray. In addition, a DA-Bead plate can transfer beads in a minute or so by uniquely designed magnet array eliminating the need of tedious and error-prone multiple pipetting steps. The reduction of samples and beads consumption by 5 – 10 fold has been enabled by efficient incubation through intermittent shaking and effective wash through laminar flow on individual beads on a wall-less plate. The consumption of only 10-20 microliters of sample and 10 microliters of beads per plate droplet makes DA-Bead plates ideal for precious samples such as mouse sera and human tears.
Singulex assay achieves ultra-high sensitivity by capturing all the target analyte available in 100 microliters of a sample and detect by Single Molecule Counting (SMC™) technology. How is it possible to achieve comparable sensitivity when the sample volume is reduced to 10 – 20 microliters instead of 100 microliters?
It is true that the sample volume of 10 – 20 microliters will have 5 – 10 fold fewer target analyte than 100 microliters required for a traditional plate method. DropArray technology has overcome this inherent, grand challenge by improving the efficiency of binding and minimizing non-specific binding and demonstrated comparable sensitivity to a traditional plate method. When a Singulex assay is run side-by-side by a traditional plate and DA-Bead plate, signal intensity is smaller on a DA-Bead plate as expected. However, the background signal is reduced accordingly producing a comparable LLOQ and signal ratios enabling same quantification between calibrators and samples.
If a DA-Bead plate can miniaturize a Singulex assay by 5 – 10 fold, why is it not possible on a traditional plate? Can I try the same reduction of a sample and bead reagent while maintaining the same LLOQ and quantification?
The simple answer is NO. Curiox’s team has met a number of Singulex assay users who have tried to reduce the sample consumption as the use of 100 microliters of a sample per well is too much for their precious animal and human samples. A DA-Bead plate has unique incubation and wash methods that improves binding efficiency by increasing the number of specific binding and minimizing non-specific binding thanks to the innovative wall-less feature of a plate. A traditional 96-well microplate with walls would not be able to achieve such improvement due to the presence of a physical wall.
I have an in-house developed Singulex assay. Would it work as effectively as a commercial, off-the-shelf Singulex assay by DA-Bead plate?
Yes. An in-house developed Singulex assay works equally well as a commercial kit assay by DA-Bead plate. It may need 1-2 days of optimization mostly optimizing the number of wash cycles and amount of beads.
Does the whole-plate washing of a DropArray DA-Bead plate cause cross-contamination between droplets as Singulex assay is ultra-sensitive?
No. A number of tests performed by Curiox’s R&D team and DropArray users have demonstrated no detectable cross-contamination between droplets. In the perspective of cross-contamination, there are two possible pathways: cross-contamination of beads or samples. The cross-contamination of beads has been tested 1) by dispensing an excessive number of beads in alternate drop on a plate with blank drops on the rest of the plate and 2) by dispensing ~10 beads per drop onto an entire plate and physically count. In both cases, beads were observed to stay where they are after 10x wash cycles, suggesting that there is no bead loss nor cross-contamination between drops. The cross-contamination of samples has been tested by running a Singulex assay with an analyte of an extremely high concentration on one spot and a blank buffer on the rest of a plate. In both cases, the blank spots right next to the control spots showed same background signals as those farthest from control spots, suggesting no detectable cross-contamination of reagents to neighboring spots.
A simple answer is No. More specifically, there are two areas the workflow may be different from a traditional plate method. First, a DA-Bead plate may need a sample incubation step performed twice where a second sample incubation step needs another fresh sample of 10 microliters after the first sample incubation step. Such repeat of a sample incubation step has shown enriching a target analyte effectively. Second, a DA-Bead plate replaces a cumbersome and error-prone beads transfer step by pipetting with a transfer step by a unique magnet array simplifying the step. Otherwise, the entire workflow of a Singulex assay on a DA-Bead plate is identical to that of a conventional microtiter plate. The DA plate replaces the traditional microplate and the DropArray Washing Station is used during the washing steps.
DA-HCS for Cellular Assays
No. The DropArray platform allows seamless use on any conventional dispenser, gripper, stacker, reader or imager.
The DropArray DA-HCS plate can be used with a low confluence of 80 cells per mm2 to a very high confluence of 630 cells per mm2. For first time users, we recommend 250 cells per mm2. The cell growth rate on DA-HCS plates is no different compared to a conventional microplate.
Depending on cell-line and assay requirements, cells can survive up to two weeks without media exchange. Media can be aspirated manually or washed off using DropArray HT/LT Washing Stations before applying fresh media.
DropArray DA-HCS plates are tissue culture-compatible. Like a conventional tissue culture plate, DA-HCS plates can be easily coated with DNA, poly-lysine, Collagen, fibronectin, silane-compatible substrates, or any other cell culture coatings. In addition, pre-coated plates for different cellular assays are available.