Plate reader: Difference between revisions

Revision as of 21:21, 18 January 2022

Revision as of 02:10, 4 January 2023

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{{more citations needed|date=April 2012}}

'''Plate readers''', also known as '''microplate readers''' or '''microplate photometers''', are instruments which are used to detect [[biology|biological]], [[chemistry|chemical]] or [[physics|physical]] events of samples in [[microtiter plate]]s. They are widely used in research, [[drug discovery]],<ref>{{Cite journal|lastlast1=Neves|firstfirst1=Bruno Junior|last2=Agnes|first2=Jonathan Paulo|last3=Gomes|first3=Marcelo do Nascimento|last4=Henriques Donza|first4=Marcio Roberto|last5=Gonçalves|first5=Rosângela Mayer|last6=Delgobo|first6=Marina|last7=Ribeiro de Souza Neto|first7=Lauro|last8=Senger|first8=Mario Roberto|last9=Silva-Junior|first9=Floriano Paes|last10=Ferreira|first10=Sabrina Baptista|last11=Zanotto-Filho|first11=Alfeu|date=2020-03-01|title=Efficient identification of novel anti-glioma lead compounds by machine learning models|url=http://www.sciencedirect.com/science/article/pii/S022352341931133X|journal=European Journal of Medicinal Chemistry|language=en|volume=189|pages=111981|doi=10.1016/j.ejmech.2019.111981|pmid=31978780 |s2cid=210892159 |issn=0223-5234}}</ref> bioassay validation, quality control and manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 1-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is 96-well (8 by 12 matrix) with a typical reaction volume between 100 and 200 µL per well. Higher density microplates (384- or 1536-well microplates) are typically used for screening applications, when throughput (number of samples per day processed) and assay cost per sample become critical parameters, with a typical assay volume between 5 and 50 µL per well. Common detection modes for microplate assays are absorbance, [[fluorescence]] intensity, [[luminescence]], [[Time-resolved spectroscopy#Time-resolved fluorescence spectroscopy|time-resolved fluorescence]], and [[fluorescence polarization]].

==Methods==

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===Fluorescence===

Fluorescence intensity detection has developed very broadly in the microplate format over the last two decades. The range of applications is much broader than when using absorbance detection, but the instrumentation is usually more expensive. In this type of instrumentation, a first optical system (excitation system) illuminates the sample using a specific wavelength (selected by an optical filter, or a monochromator). As a result of the illumination, the sample emits light (it fluoresces) and a second optical system (emission system) collects the emitted light, separates it from the excitation light (using a filter or monochromator system), and measures the signal using a light detector such as a [[photomultiplier]] tube (PMT). The advantages of fluorescence detection over absorbance detection are sensitivity, as well as application range, given the wide selection of fluorescent labels available today. For example, a technique known as [[calcium imaging]] measures the fluorescence intensity of [[calcium-sensitive dyes]] to assess intracellular calcium levels.{{citation needed|date=May 2020}}<ref>{{Cite journal|lastlast1=Lin|firstfirst1=Kedan|last2=Sadée|first2=Wolfgang|last3=Mark Quillan|first3=J.|date=February 1999|title=Rapid Measurements of Intracellular Calcium Using a Fluorescence Plate Reader|url=https://www.future-science.com/doi/10.2144/99262rr02|journal=BioTechniques|language=en|volume=26|issue=2|pages=318–326|doi=10.2144/99262rr02|pmid=10023544 |issn=0736-6205|doi-access=free}}</ref>

===Luminescence===

Luminescence is the result of a chemical or biochemical reaction. Luminescence detection is simpler optically than fluorescence detection because luminescence does not require a light source for excitation or optics for selecting discrete excitation wavelengths. A typical luminescence optical system consists of a light-tight reading chamber and a [[Photomultiplier|PMT]] detector. Some plate readers use an Analog PMT detector while others have a [[photon counting]] PMT detector. Photon Counting is widely accepted as the most sensitive means of detecting luminescence. Some plate readers offer filter wheel or tunable wavelength monochromator optical systems for selecting specific luminescent wavelengths. The ability to select multiple wavelengths, or even wavelength ranges, allows for detection of assays that contain multiple luminescent reporter enzymes, the development of new luminescence assays, as well as a means to optimize the signal to noise ratio.{{citation needed|date=May 2020}}

Common applications include [[luciferase]] -based gene expression assays, as well as cell viability, cytotoxicity, and biorhythm assays based on the luminescent detection of [[Adenosine triphosphate|ATP]].<ref>{{Cite journal|lastlast1=Lin|firstfirst1=Kedan|last2=Sadée|first2=Wolfgang|last3=Mark Quillan|first3=J.|date=1999-02-01|title=Rapid Measurements of Intracellular Calcium Using a Fluorescence Plate Reader|url=https://www.future-science.com/doi/10.2144/99262rr02|journal=BioTechniques|volume=26|issue=2|pages=318–326|doi=10.2144/99262rr02|pmid=10023544 |issn=0736-6205|doi-access=free}}</ref>

===Time-resolved fluorescence (TRF)===

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*Cell toxicity, proliferation, and viability

*ATP quantification

*[[Immunoassays]]<ref>{{Cite journal|lastlast1=Ashour|firstfirst1=Mohamed-Bassem A.|last2=Gee|first2=Shirley J.|last3=Hammock|first3=Bruce D.|date=1987-11-01|title=Use of a 96-well microplate reader for measuring routine enzyme activities|url=httphttps://wwwdx.sciencedirectdoi.comorg/science10.1016/article/pii/00032697879058590003-2697%2887%2990585-9|journal=Analytical Biochemistry|language=en|volume=166|issue=2|pages=353–360|doi=10.1016/0003-2697(87)90585-9|issn=0003-2697}}</ref>

*[[High throughput screening]] of compounds and targets in drug discovery

*Bead-Based Epitope Assay<ref>{{cite journal |last1=Suprun |first1=Maria |last2=Getts |first2=Robert |last3=Raghunathan |first3=Rohit |last4=Grishina |first4=Galina |last5=Witmer |first5=Marc |last6=Gimenez |first6=Gustavo |last7=Sampson |first7=Hugh A. |last8=Suárez-Fariñas |first8=Mayte |title=Novel Bead-Based Epitope Assay is a sensitive and reliable tool for profiling epitope-specific antibody repertoire in food allergy |journal=Scientific Reports |date=5 December 2019 |volume=9 |issue=1 |page=18425 |doi=10.1038/s41598-019-54868-7 |pmid=31804555 |pmc=6895130 |bibcode=2019NatSR...918425S }}</ref>