Frequently Asked Questions

 

General
What are the major differences between the DropArray Microplate and a conventional microplate?

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 standard. Using standard microplate pipetting devices, 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 washing buffers added in bulk during wash steps of the assay. Unlike a standard microplate that requires dispensing and aspiration from each well, bulk liquids like washing 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. Evaporation is controlled using an Evaporation Control Lid which provides a humidified environment and minimizes evaporation of the droplets.

What are the distinct advantages of DropArray Microplates over conventional microplates?

DropArray Microplates enable simple and easy miniaturization of assays at 2-4 microliters for the 384-well format and 5-20 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 conventional microplates. Furthermore, the wall-less feature allows extremely gentle washing by liquid exchange, which is different from the harsh, convectionbased washing performed by a microplate washer with an array of nozzles.

Luminex® Assays
What are the benefits of using DropArray DA-Bead plates for running Luminex assays such as Milliplex®, Bio-Plex® and Procartaplex® assays?

A DA-Bead plate improves sensitivity, reproducibility, detectability, and reduces the consumption of both samples and beads to 5 microliters each. The improvement of sensitivity, reproducibility, and detectability 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 standard 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 pipet 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 lower 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 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. On the contrary, the sensitivity improves due to unique incubation and washing procedure on a DA-Bead plate. In a Luminex assay, the ratio of sample volume to the number of beads is determined at 25 microliters of sample for 2,000~2,500 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 the superior washing and minimizing background signal, the sensitivity is at least equivalent or improved compared to the microtiter plate method.

What are the features of DA-Bead plate that enables better sensitivity and reproducibility?

The features that enable better sensitivity and 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.

How does Luminex assay run on DA-Bead plate show better sensitivity with samples of body fluids?

There are three reasons for the better performance in biological fluids. First, the data on a DA plate are much less affected by matrix effect and non-specific binding and show good linearity while the data on a standard microtiter plate show substantial deviation from linearity. Second, the CV is smaller on a DA-Bead plate allowing better and reliable quantification even at low concentrations. Last, eliminating background signal is important and since the DA-Bead plate enables better washing, background signal is reduced.

Why do Luminex assay run on a DA-Bead plate show better reproducibility?

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. 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 better 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 microtiter plate showed minimal matrix effect.

If the magnetic retention of beads is partly responsible for the better data, can I use the method for my Luminex assay on microtiter plate?

Based on preliminary data from Curiox’s team, this method does not work on microtiter plate because there is another critical factor missing for microtiter plate – 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 hinder thorough mixing and binding when beads are retained on a surface.

I am using high-plex assay of 40-60plex. 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. Empirically, beads of up to 200plex would form a single layer on DA-Bead plate.

I am using CBA, Singulex or Quanterix assays, which use beads for immunoassay. Would my assay benefit by using DA-Bead plate?

Curiox’s team has not run these assays yet on the platforms mentioned above. However, theoretically the DA plate technology could work with these platforms.

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 2,000~2,500 beads per analyte. A 5 microliter bead solution contains ~400-500 beads per analyte, which is still excessive for the number of beads required for reading.

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. 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.

Is it easy to use a DA-Bead plate, particularly in dispensing? Doesn’t it require special skills as it has no walls?

A DA-Bead plate is as easy to use as a conventional microtiter plate 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, a DA-Bead plate is much easier to use than a conventional microplate. As a matter of fact, even 2 microliters of reagents are distinctly visible as a droplet on a plate.

Does a DA-Bead plate change my current Luminex assay workflow on a conventional microplate?

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 microtiter plate and the DropArray Washing Station is used during the washing steps. You can watch a video of the entire assay run on a DropArray DA-Bead plate at http://www.curiox.com/videos.html.

How much wash buffer and washing time are needed to wash plates on the DropArray Washing Station?

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.

Are DropArray Washing Stations automation-friendly?

Yes. The DropArray HT Washing Station (HT200) has a PC-HT200 RS232C port for communication with automation controllers.

Cellular Assays
Do we need any specialized dispensers or imaging systems?

No. The DropArray platform allows seamless use on any conventional dispenser, gripper, stacker, reader or imager.

What are the cell growth parameters on a 384-well DropArray DA-Cell plate?

The DropArray DA-Cell 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-Cell plates is no different compared to a conventional microplate.

How long can the cells survive on DropArray DA-Cell plates? How do I perform media exchange?

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.

Do the DA-Cell plates come coated?

DropArray DA-Cell plates are tissue culture-compatible. Like a conventional tissue culture plate, DA-Cell 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.

Milliplex, Bio-Plex, Procartaplex and Luminex are either trademarks or registered trademarks of eBioscience and Luminex Corporation respectively in the US and/or other countries. Curiox DA-Bead plates are not endorsed by the Luminex Corporation.