Product Survey: Microplate Readers
by Harald Zähringer, Labtimes 05/2016
Assay detection in microplate readers is still dominated by light-based approaches. However, label-free plate readers based on resonant waveguide gratings offer new possibilities to creative researchers.
Light is reflected at the resonant waveguide gratings of this biosensor chip. The same phenomenon is utilised in label-free microplate readers to detect changes in surface mass. Photo: Greg Pluta, University of Illinois at Urbana-Champaign
Microplate (plate) readers are the archetypical jack-of-all-trades detection instruments in life science labs. From protein and nucleic acid quantifications, to UV-Vis absorbance, fluorescence and luminescence measurements, analysis of ELISAs, complex FRET, BRET, fluorescence-polarisation, Alpha-Screen or Time-Resolved fluorescence experiments: microplate readers can do it all.
Light-based microplate readers may be categorised into single-mode instruments, dedicated to only one measuring mode, usually absorption, fluorescence or luminescence, and multi-mode readers, integrating several detection technologies into one instrument, enabling researchers to rapidly switch between different assay formats. Multi-mode readers are equipped with different optical systems, each tailored to the special needs of the respective detection mode. At the heart of the optical (excitation) systems are filters and/or monochromators, separating white (multichromatic) light, coming from a tungsten, LED or Xenon flash lamp, into a monochromatic light beam that is channelled via mirrors or fibres to the microplate wells.
Optical filters are cheap and let pass the desired wavelength at a defined bandwidth, with minimal signal loss and light scattering. But filters must be changed every time the operator switches to another excitation wavelength. Besides that, plate readers usually contain only a limited set of standard filters for routine applications. Researchers must buy additional filters to perform experiments requiring non-standard wavelengths. And there is one more drawback of filter-based plate readers: they are not suitable for spectral scanning of cellular assays, which may be useful to characterise, for example, unknown fluorophores.
Hence, many manufacturers rather rely on monochromators for wavelength selection or combine both, filters and monochromators, in their instruments. Monochromators are basically moveable optical grids that split white light, entering the system via a narrow entry slit, into a spectrum that is projected onto a tight exit slit. Light of the desired wavelength passes the exit slit and is subsequently conducted to the microplate well. The wavelength can be adjusted almost continuously in tiny nanometre steps by simply rotating the monochromator grid.
But there’s no such thing as a free lunch in optics – the flexibility of the monochromator grid is paid for with higher light scattering, leading to a lower signal-to-noise ratio. Plate reader manufacturers compensate the increased light scattering with a simple but effective trick: they connect two monochromator systems in series. The split beam, passing the exit slit of the first monochromator, is directly channelled into the entry slit of the second monochromator to keep out unwanted stray light. The very same principle is also applied on the emission side of monochromator readers: fluorescence or luminescence light irradiated from the microplate wells passes two monochromator systems, before entering a scientific camera or a photomultiplier tube. Hence, plate readers with two double monochromator systems are often dubbed quadrupole plate readers.
Monochromators may also be constructed with Linear Variable Filters (LVF), which are basically wedged filters with linearly varying spectral properties. LV longwave pass filters allow transmission of long wavelengths, LV shortwave pass filters let short wavelengths pass. Arranging both filter types in series leads to a band-pass filter, which may be tuned from 320 nm to 850 nm by simply moving the filters linearly against each other. LVF monochromators combine the spectral advantages of filters with the flexibility of moveable grids. And there’s another plus to LVFs that comes in very handy when flexible and sensitive measurements are needed: the bandwidth is adjustable in nanometre steps, enabling bandwidths ranging from a few nanometres to 100 nanometres.
Signal detection in microplate readers is almost exclusively based on light phenomena, originating from fluorescent or luminescent-labelled molecules. The only exceptions are label-free microplate readers, utilising resonant waveguide gratings (RWG) for measuring refractive index changes at the surface of a special microplate. Sounds a bit weird but the basic idea of RWG readers is simple. At the heart of a RWG biosensor is a periodic rectangular grating, embedded into a waveguide film that covers the surface of a microplate. The plate is angularly illuminated (usually from below) with polarised light. The light of a specific wavelength couples into the planar waveguide and shortly propagates along the grating before it is reflected. The coupling efficiency of the resonant wavelength into the RWG strictly depends on the local refraction index near the surface of the biosensor. Binding of molecules or cells to the surface (increasing the surface mass) alters the reflection index leading to a shift in resonance wavelength, which is detected in current RWG readers by elaborated sensors.
Recently, however, researchers centred around Martina Gerken’s Nanophotonics group at the University of Kiel, Germany, RWG pioneer Corning and the start-up company Byosens, Hamburg, came up with a new, “simple” RWG sensor technique that has been implemented in a portable mini-RWG-reader (Nazirizadeh et al., Scientific Reports, 6:24685). The mini-reader’s biosensor utilises changes in the intensity of the resonant wavelength, instead of wavelength shifts for detection. Alterations in resonance intensity have not been used in previous RWG sensors because of their ambiguous relation to refraction index changes. But this is not true for resonances close to the substrate cut-off wavelength: cut-off resonance wavelength intensity strictly depends on reflection index changes.
Measuring the resonance intensity is simply done with a photo diode that captures a LED beam, reflected at the waveguide grating. The intensity-based RWG sensor covers the RWG microplate similar to a lid and enables microplate-sized, label-free readers that can be integrated into robotic systems. But it’s not only the small size and the high-throughput possibilities that makes the mini-reader an attractive instrument for life scientists. The intensity-based biosensor may also inspire researchers from different fields to come up with their own ideas for label-free microplate reader applications.
First published in Labtimes 05/2016. We give no guarantee and assume no liability for article and PDF-download.
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