Microplate Reader - Working Parameters And Detection Methods

In the scientific world, many instruments work in the process of observations of biological specimens.  One such instrument is the microplate readers that work in the direction of detection of chemical, biological, and physical events of specimens in microtiter plates. These plates are found in the form of microtiters to enhance the observations made by them.  Microplate readers find its use in research, bioassay validations, drug discovery, academic organizations,  quality control, and manufacturing processes in pharmaceutical and biotechnology firms.  These industries have employed the microplate reader for giving out good results from the biological specimens.

In the microplate reader, the sample reactions are assayed in the range of 1- 1536 well format microtiter plates in a biological research lab. It is seen that the microplate reader formats are used in academic research laboratories and clinical diagnostic laboratories for the observations.   The most common well range is the 96- well type which is 8/ 12 matrix of the microplate reader.   This also has a typical reaction volume between 100 and 200-micron liters per well in the elisa plate reader. Elisa tests are also done with the microplate reader for cancer research as well.  It is believed that the best microplate readers with small reaction rates are ideal. However, the higher density microplate readers are also there with 384 -1536 wells.   This higher elisa reader is also used for the screening processes of all types of biological specimens.  Elisa reader price is somewhat expensive than simpler readers. 

What Is A Microplate Reader?

A micro-plate reader is a device of lab importance used to analyze biological, physical, and chemical reactions, properties of all biological specimens within the wells. The plates consist of numerous wells which have the specimens for the analysis process.  These are also called analytes which are assessed with the help of a microplate reader in a lab.  In all wells of an elisa plate reader, different reactions take place which helps in the separate assessment of all types of analytes.  Further, this separation helps in the progression of biochemical processes which are transformed into optical signals.  These signals are detected by the microplate reader and thus, quantifies the data from the specimen.

Scientists and researchers of life sciences and pharmaceuticals industries are striving to make the process more efficient and proficient. The microplates are used to analyze the specimens present in the wells by handling them properly in the research lab.   A microplate reader helps all scientists and researchers to reduce the operational time and also save the reagent costs. This allows the researchers to dedicate more time to the data analysis and generation of good results. The typical essay is between the 5 and 50-micron liter per well in the microplate reader.   

Detection Methods Or Detection Methods Used by An Microplate Reader

Following are the common  detection methods of microplate readers:

• Absorbance
• Fluorescence Intensity
• Time-Resolved Fluorescence
•  Fluorescence Polarization
• Luminescence

Let us take a closer look at the most used methods of detection by the multimode plate reader.

1.  Absorbance

Absorbance detection by microplate readers has been available in the scientific world for a long time. For more than 3 decades, the detection processes are implemented in the world. They are used for various assays like ELISA, protein assays, and nucleic acids assays. The enzyme quantification or enzymatic assays are also done by this microplate reader detection method.  

In this process of detection, a  light source illuminates the biological specimen,  using a specific wavelength. Here the wavelength is selected by an optical filter or with a monochromator.  A  light detector located on the other side of the well in the plate measures how much of the initial light is transmitted through the biological sample.  The amount of transmitted light is typically related to the concentration of the molecule of interest present within the wells.  Several other conventional colorimetric analyses have been introduced into the wells of an elisa microplate reader to analyze the specimens correctly. It is further miniaturized to function quantitatively in a  microplate reader with performance suitable for biological research purposes.

Examples of such analyses used in the microplate reader absorbance are ammonium, urea, orthophosphate, nitrate, nitrite, iron, etc.  In the analyses of all these, the colorimetric chemistries are also used for the perfect analysis of samples.

2. Fluorescence Intensity Detection

Fluorescence intensity detection has developed extensively in microplate format over the last two decades of scientific research. The range of applications of fluorescence intensity detection is much broader than the microplate absorbance detection method.  However, the instrumentation for fluorescent detection is usually more expensive. In this type of microplate reader, the  instrumentation consists of :

• First optical system- This excitation System illuminates the biological sample using a specific wavelength of light. It is usually selected by an optical filter or monochromator.
• The illumination system –As the light rays are transmitted through the specimen,  it emits light which is called fluorescence.
• Second optical system – The emission system of the microplate reader, which collects the emitted light and separates it from the excitation light. This separation is done by using a monochromator system. It also measures the signal using a light detector, called a photomultiplier tube (PMT).

The main advantages of using fluorescence detection over microplate reader  absorbance detection are :

• Sensitivity
• The application range has given the wide selection of fluorescent labels available.

An example of this fluorescence technique is calcium imaging which measures the fluorescence intensity of calcium dyes used in the analysis process.

3. Time – Resolved Fluorescence ( TFR)

The time–resolved fluorescence measurement is somewhat similar to the fluorescence intensity measurement by the multi-well reader. The only difference between the two is the timing of the excitation and measurement process.

When measuring the fluorescence intensity of the sample, the excitation and emission of photons processes are simultaneous in nature.  The light emitted by the biological sample is measured while the excitation process takes place in the plate reader. 

The fluorescence optical system depends on the use of specific fluorescent molecules,  known as lanthanides.  These molecules have the unusual property of emitting over long periods of time in the reader which are measured in milliseconds after the concluding in excitation.   In the process of fluorescence intensity, the standard fluorescent dye called fluorescein which emits within a few nanoseconds of being excited the process.  This all results in the excitation of lanthanides using a pulsed light source in the microplate reader.  After the excitation process, the measurements are taken by the researchers.

The main usage of TRF is in the area of drug screening applications in the pharmaceutical industries in the form of time resolved fluorescence energy transfer.

4. Luminescence Detection Method

The luminescence detection method is the result of a chemical and biochemical reaction within the wells of the microplate reader. Luminescence detection is a simpler optical phenomenon than the above detection methods. It does not require a light source for the excitation of light rays and also does not require the selection of discrete excitation wavelengths.

A usual luminescence optical system consists of:

• A  light-tight reading chamber and
• A PMT detector

Some microplate readers use an Analog PMT detector for assessing the result. While the other microplate readers have a photon-counting PMT detector.  his counting photon is widely accepted as the most sensitive means of detecting luminescence within the wells of the microplate reader.

It allows the detection of assays that contain multiple luminescent marking enzymes and the development of new luminescence assays.

Conclusion

Microplate readers are the laboratory instruments that are used to detect the biological, chemical, and biological aspects of the biological specimens. The analysis takes place separately within the wells of the microplate readers.