Microplate reader - Do you know about it?
Do you believe in "light"?
Light is an electromagnetic wave. Light with a wavelength of 100nm-400nm is called ultraviolet light, light between 400nm-780nm can be observed by the human eye, and light greater than 780nm is called infrared light. The reason people can see colors is that light shines on an object and is reflected back by the object. The reason green plants are green is that they absorb the red spectrum of light. The principle of measurement by a microplate reader is to detect the absorbance value of the measured substance at a specific wavelength.
"Light" and Microplate Readers
A microplate reader, also known as an enzyme-linked immunosorbent assay (ELISA) detector, is used to measure the absorbance after an ELISA reaction. It mainly consists of an optical system and a signal acquisition system. Its working principle is as follows:
Light waves emitted by the source lamp pass through a filter or monochromator to become a beam of monochromatic light. Part of this monochromatic light is absorbed by the sample, while the other part passes through the sample and irradiates the photoelectric detector.
The photoelectric detector converts the light signal into a corresponding electrical signal. After a series of signal processing steps, the signal is sent to the microprocessor for data processing and calculation, and the result is finally displayed.
What "Light" is Used for Detection?
Principle of Measurement Wavelength
When light passes through the substance being detected, the difference in energy before and after passing through is the energy absorbed by the substance. At a specific wavelength, the concentration of the same substance has a quantitative relationship with the absorbed energy.
The detection unit is expressed as the OD value. OD is the abbreviation for optical density, which represents the optical density absorbed by the substance being detected. OD = log(1/trans), where trans is the transmittance value of the substance. According to the Bouguer-Lambert-Beer law, the relationship between the OD value and light intensity is as follows:
E = OD = log(I₀/I)
Here, E represents the absorbed optical density, I₀ is the light intensity before the substance, and I is the light intensity after passing through the substance.
The OD value is calculated using the following formula:
E = OD = C × D × E
Where: C is the concentration of the substance; D is the thickness of the substance; E is the molar extinction coefficient.
Each substance has its specific wavelength at which it can absorb the most light energy. If another wavelength range is selected, it may lead to inaccurate detection results. Therefore, when measuring a substance, we choose a specific wavelength for detection, known as the measurement wavelength.
However, each substance also has some non-specific absorption of light energy. To eliminate this non-specific absorption, we select a reference wavelength to remove this inaccuracy. At the reference wavelength, the light absorption by the substance is minimal. The difference in absorbance between the detection wavelength and the reference wavelength can eliminate non-specific absorption.
Commonly Used Wavelengths
Generally, the measurement wavelength of a microplate reader ranges between 400–750nm or 800nm, which fully meets the requirements for colorimetric determination in ELISA.
Currently, the enzymes commonly used for labeling in ELISA kits in China are horseradish peroxidase (HRP), and the substrates are usually tetramethylbenzidine (TMB) and ortho-phenylenediamine (OPD). In the presence of hydrogen peroxide solution, they are oxidized by HRP into diaminobenzidine (DAB) and biphenylquinone, respectively.
When the pH is around 5.0, DAB has maximum absorption at a wavelength of 450nm.
When the pH drops to 4.0, the maximum absorption wavelength shifts to 492nm, and the molar extinction coefficient increases, deepening the color. Therefore, strong acids such as sulfuric acid or hydrochloric acid are often used to terminate the reaction.
The oxidation product of TMB, biphenylquinone, has the maximum extinction coefficient at a wavelength of 450nm. If the amount of HRP is small, and H₂O₂ and TMB are in excess, a blue cation radical is formed.
Lowering the pH can convert the blue cation radical into yellow biphenylquinone. Using sulfuric acid as a termination agent can stabilize the product for 90 minutes.
The two wavelengths of 450nm and 492nm are the most commonly used in ELISA measurements. Considering the need for dual-wavelength colorimetry, filter lenses with wavelengths of 630nm and 405nm are also required.
Single-Wavelength/Dual-Wavelength Detection
Microplate readers have both single-wavelength and dual-wavelength detection functions. Sometimes users are unsure when to use single or dual-wavelength detection.
"Single-wavelength" refers to colorimetric measurement using one wavelength with maximum absorption for the color development, such as 450nm or 492nm.
"Dual-wavelength" refers to colorimetric measurement using not only the wavelength with maximum absorption for the color development (e.g., 450nm or 492nm) but also a wavelength insensitive to the specific color development, such as 630nm. The absorbance finally printed by the microplate reader is the difference between the two.
Single-Wavelength/Dual-Wavelength Detection
Microplate readers have both single-wavelength and dual-wavelength detection functions. Sometimes users are unsure when to use single or dual-wavelength detection.
"Single-wavelength" refers to colorimetric measurement using one wavelength with maximum absorption for the color development, such as 450nm or 492nm.
"Dual-wavelength" refers to colorimetric measurement using not only the wavelength with maximum absorption for the color development (e.g., 450nm or 492nm) but also a wavelength insensitive to the specific color development, such as 630nm. The absorbance finally printed by the microplate reader is the difference between the two.
The absorbance obtained at 630nm is non-specific and comes from absorption caused by factors such as fingerprints, dust, and dirt on the plate wells. Therefore, in ELISA colorimetric determination, it is best to use dual-wavelength detection, and there is no need to set a blank well.
Absorbance Measurement Range
Generally, the absorbance measurement range of a microplate reader between 0–2.5 can meet the requirements of ELISA measurements. Early microplate readers could typically measure absorbance in the range of 0–2.5, but now the range has basically been expanded to 3.5 or higher while maintaining good precision and linearity. Therefore, there is no need to deliberately pursue a large absorbance range for microplate readers; the key is to examine the linearity and precision within a certain absorbance range.
Detection Speed
The detection speed of a microplate reader refers to the time required to complete the colorimetric measurement. As the saying goes, "In martial arts, speed is the key to invincibility." Fast detection speed helps improve detection precision by avoiding differences in absorbance between wells due to variations in measurement time. Currently, the detection speeds of common microplate readers on the market are very fast, usually within a few seconds.
Plate Shaking Function
The plate shaking function of a microplate reader refers to oscillating and mixing the ELISA plate wells before colorimetric measurement to ensure uniform color within the wells. Currently, common microplate readers on the market have a plate shaking function, but the shaking methods may differ. Some can be adjusted arbitrarily in terms of up-down, left-right, or rotational shaking methods and oscillation amplitudes. When using a microplate reader with a plate shaking function, after adding the stop solution to complete the color development reaction in ELISA, you can directly place it into the microplate reader for measurement without oscillating or mixing.
Incubation Function
The incubation function refers to the microplate reader's ability to automatically and precisely control the internal temperature as required, allowing the incubation process of the microplate strips in ELISA measurements to be completed inside the instrument without the need for an external incubator.
The incubation function is only an additional feature of a microplate reader. Its presence or absence does not indicate the grade of the microplate reader or the quality of the instrument. Whether a laboratory chooses this function depends on the laboratory's conditions and needs.
Detection Items
▶Clinical Testing Items
★Infection Immunology Testing: Hepatitis B五项 (HBV), Hepatitis A Antibody (HAV), Hepatitis E Antibody (HEV), Hepatitis C Antibody (HCV), Immunoglobulin (IgG), HIV Antibody (HIV), Treponema pallidum Antibody...
★Hematological Immunology Testing: Immunoglobulin A/G/M/E/D, Immunoglobulin Light Chain κ/λ, Complement C3/C4, CRP, Rheumatoid Factor, Prealbumin, Ceruloplasmin, Haptoglobin, β2-Microglobulin, Transferrin, Acid Glycoprotein, High-Sensitivity CRP, Serum Amyloid A, γ-Trace Protein, Circulating Immune Complex, ANA, ENA...
★Allergen Testing: Total Serum IgE, Specific IgE and Screening Tests, ECP Test...
★Tumor Marker Testing: Carcinoembryonic Antigen (CEA), Alpha-Fetoprotein (AFP), Prostate-Specific Antigen (PSA), Carbohydrate Antigen (CA199), Carbohydrate Antigen (CA125), Carbohydrate Antigen (CA153), Squamous Cell Carcinoma Antigen (SCC)...
★Reproductive Medicine Testing: Toxoplasma, Rubella Virus, Cytomegalovirus, Herpes Simplex Virus Type I and II, Anti-Sperm Antibody, Anti-Endometrial Antibody, Anti-Ovarian Antibody, Anti-Trophoblast Cell Antibody, Anti-hCG Antibody...
▶Food Safety Testing Items
★Allergens: Food;
★Melamine: Agricultural Products and Food;
★Dioxins: Agricultural Products and Food, Environmental Samples;
★Veterinary Drug Residues
★Antibiotics: Agricultural Products and Food;
★Clenbuterol/Lean Meat Powder: Urine, Hair, Meat, Milk, Feed of Poultry and Livestock, etc.;
★Pesticide Residues: Agricultural Products, Food, Water, Soil, etc.
★Insecticides, Fungicides, Herbicides, Plant/Insect Growth Regulators;
★Biotoxins: Food and Feed
★Aflatoxin; Shellfish Toxin; Vomitoxin; Aflatoxin B1; Zearalenone; Ochratoxin A.
▶Exit-Entry Inspection and Quarantine Items
★Health Quarantine: Hepatitis C, HIV, Syphilis Antibody Testing for Entry-Exit Personnel;
★Animal CIQ: Porcine Transmissible Gastroenteritis Virus (Biotin Labeled 414 nm), Porcine Respiratory Coronavirus (Biotin Labeled 414 nm), Porcine Epidemic Diarrhea, Infectious Bursal Disease of Chickens (490 nm), Infectious Laryngotracheitis of Chickens, Infectious Bovine Rhinotracheitis (450 nm), etc. for Entry-Exit Animal Virus Detection;
★Plant CIQ: Tomato Black Ring Virus Detection, Cowpea Severe Mosaic Virus (Alkaline Phosphatase Labeled 405 nm).
▶Cell Proliferation and Cytotoxicity Testing
★MTT Method:
By measuring the absorbance at a wavelength of 570nm, the number of living cells can be indirectly reflected. The faster and more the cells proliferate, the higher the absorbance value; the greater the cytotoxicity, the lower the absorbance value.
★CCK-8:
Measure absorbance at 450nm: It is recommended to use dual-wavelength measurement, with a detection wavelength of 450-490nm and a reference wavelength of 600-650nm.
▶Protein Concentration Determination (Colorimetric Method)
★BCA Method: 562 nm;
★Bradford Method: 595 nm;
★660nm Protein Analysis Quantitative Reagent: 660nm.
▶Endotoxin Testing
★Dynamic Chromogenic Method (Microtechnology): 405nm.
▶Enzyme Activity测定
Principle: The detection of enzyme activity is based on the consumption or production of the substrate NADH, which can be detected to indirectly quantify enzyme activity.
Detection Method: Under incubation at 37°C, enzyme activity is determined using kinetic detection methods at 340nm.
★Specific Applications: Alanine Aminotransferase (ALT) Testing (Liver Function), Urea Nitrogen Testing (Urease-NADH Method) (Kidney Function), Glucose-6-Phosphate Dehydrogenase Testing (Hemolytic Disease), Hexokinase (Serum Glucose).
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