Hybrid Technology™ provides both Spectral Filter and Monochromator-based Detection in Multi-Mode Microplate Readers

The Multi-Mode Microplate Reader is a ubiquitous and essential laboratory tool for qualitative and quantitative analysis. Providing multiple detection modes allows for the use of a wide variety of quantitative assays from colorimetric ELISAs to small molecule drug screening assays using proximity-based technologies that require no washing steps, such as time-resolved fluorescence resonance energy transfer (TR-FRET). Typically, these assays require high sensitivity and in the case of TR-FRET screening assays, rapid switching of detection wavelengths to generate signals. The most optimal optical components for providing this need for speed and sensitivity are spectral bandpass filters. The main limitations to the use of spectral bandpass filters are that one typically requires a unique set of filters optimized for each fluorophore used and one cannot obtain spectral scans. Monochromators using diffraction gratings circumvent these issues providing the ultimate flexibility in wavelength selection and the ability to obtain spectra. The main drawback in using monochromators is the light transmission through the optical system is much less than for spectral bandpass filters and thus the sensitivity is less.

Laboratories usually must make a choice between a filter-based or monochromator-based multimode reader. If speed and/or sensitivity are the dominant need, then a filter-based system is chosen; if flexibility and spectral scanning is required, then a monochromator-based system is acquired. If all attributes are required, then typically the laboratory must buy both. BioTek’s Synergy™ 4 with Hybrid Technology™ circumvents these issues by provided both a monochromator-based optical path for fluorescence measurements and spectral filter-based modules for assays requiring speed and sensitivity. Below is a figure depicting Synergy 4’s optical paths.

Figure 1: Hybrid Technology utilizing both monochromator- and spectral filter-based optical paths.

The table below demonstrates the main attributes of filter- and monochrmator-based readers and how Hybrid Technology encompasses all.

Does your laboratory use filter- or monochromator-based microplate readers? Do you see the benefit of Hybrid Technology?

BioTek’s Gen5™ Software Offers Expanded Panel Assay Application Capabilities

Panel Assays are routinely used in diagnostic screening to test a single sample for multiple results. Although often related by applications such as drug or infectious disease screening, panel assays can also be utilized to optimize small workflow runs of completely unrelated assays. This later format can be particularly useful in laboratories that are more research driven.

The Panel Assay feature available in BioTek’s Gen5 software family can be used to define a group of up to 8 individual protocols on a single micro plate. Each protocol is individually interpreted, but samples can be grouped to display a result for each protocol. Because Gen5 is able to communicate with all BioTek readers, panels can be defined for luminescence, fluorescence, and fluorescence polarization applications in addition to the more traditional absorbance applications. This allows virtually unlimited flexibility in customizing a panel for even the most esoteric of applications.

Within Gen5 a panel is defined beginning from the Main Menu using the File/New Panel command. A wizard type prompting sequence takes the end user through all required steps of creating the panel. At runtime, tabs are available to access each of the protocols by name allowing definition of the number of samples and any other parameters unique to the protocol before the plate is read. At the end of the plate read the data is displayed according to how the samples were chosen to be categorized for the panel. The examples below illustrate results for hypothetical drug screening panels. The matrix of the plate shows how the protocols are mapped, and how samples are reported for protocol individually. The other report shows how sample results can be categorized to group results of the sample for all protocols on the panel.

Automated Kinase Profiling using a Simple and Robust Automated Platform

Kinases continue to be one of the most important targets in today’s drug discovery efforts. Following the identification of lead compounds through screening efforts, it is important to profile these leads against other kinases within that family, as well as from other families, to ascertain potential off-target effects. Typically profiling efforts include testing multiple concentrations of lead compounds in an IC50 format. Demands on a liquid handler can include performing compound titrations, as well as dispensing multiple kinase/substrate and ATP mixes.

In this application, we used the Precision™ XS Microplate Sample Processor and Synergy MX Monochromator-Based Multi-Mode Microplate Reader, in combination with Promega’s ADP-Glo™ Kinase Assay, to demonstrate automated kinase profiling. The Precision was used to perform compound titrations, as well as dispense all kinase and ADP-Glo reaction components to the wells of the assay plates. Titrations were carried out in 96-well format, with transfer volumes ranging from 20-40 μL. Kinase reactions required three 5 μL dispenses of inhibitor/DMSO, enzyme/substrate mix, and ATP. ADP-Glo reagent dispenses were set at 15 μL, and Kinase Detection Reagent dispenses at 30 μL.

Wortmannin was used as a model lead compound profiled against a series of different kinases. This compound, being a specific inhibitor for PI3 Kinase, generates IC50 values typically in the low nM range for this kinase. At higher concentrations, this compound has also shown inhibitory effects on DNA PK. Results agreed with literature values, with IC50s for PI3 Kinase Alpha and DNA PK being 0.15 and 7.1 nM, respectively. Wortmannin showed little or no effect on the remaining protein kinases.

Where do you see kinase profiling demands going in the future? What type of instrumentation do you feel is essential for automated kinase profiling? How can BioTek help you meet your kinase profiling needs?

Modular Automation Using Stand-alone Instrumentation for ELISA

After reliability and performance, the greatest concern of many researchers when contemplating automation of their assays is the utility of the instrumentation after the project is completed. Many an automated assay system has become the laboratory equivalent of the dust collecting relics known as exercise machines in many people’s homes after the project was finished. The best solution to this dilemma is to provide a modular automation system that can be upgraded, reconfigured, or disassembled and used as stand-alone instruments as needed when project and priorities change.

Automation of biochemical reactions, particularly ELISA, can easily be accomplished by configuring a robotic system that utilizes a Caliper Twister II rotating robotic arm/gripper to move microplates to and from each station. In addition to the Twister II, a Precision XS pipetting station is used to dilute samples and transfer them to the assay plate. Plate washes are carried out using an ELx405 96-well microplate washer, while reagents are added using MicroFill or MicroFlo dispensers, each dedicated to a specific reagent. Incubations can be carried out using a number of different plate hotels and incubators from Liconic or Heraeus depending on incubation requirements, and absorbance measurements are made using a PowerWave XS microplate spectrophotometer.

An automated system with a smaller physical footprint and faster process times can be configured using an EL406 washer dispenser. The EL406 is the equivalent of a total of four instruments; an ELx405 Select CW microplate washer, a MicroFlo dispenser, and two MicroFill dispensers. This configuration, while slightly less flexible, provides an increase in overall throughput as a result of less robotic movements of the microplate.

What types of ELISA reactions does your laboratory run? What sort of plate throughput-demands are required? Do you need elevated temperature or shaking incubations for your assays? What method of detection is used with your assay?

Schematic drawing with top view and side view of a robotic ELISA system.