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Let’s start with a story. In 1500 A.D., doctors found the urine of diabetics can attract ants. This method of detecting diabetes is considered to be the origin of POCT.
So, what is POCT? POCT refers to point-of-care testing. It is a segment of IVD (in-vitro diagnostics). As the name suggests, testing is carried out next to patient. Below image shows one type of POCT testing.
Contrast to conventional diagnostic instrument, POCT device has the following advantages.
- Quick turn around time. Usually it takes 3 to 15 minutes to obtain results.
- Portable design. Some devices are even handheld.
- Whole blood testing and several drops’ volume. Microfluidic based device need only one drop of blood (10 µL).
- Friendly to non-professionals. Very simple operation and easy calibration & quality control process.
- Single and ready to use cartridge, which avoids cross-contamination and reagent waste.
- Much lower cost.
|1||Laboratory||Hepatitis, Influenza, CRP, PCT testing, etc.|
|2||ICU/ CCU/ Outpatient/ Oncology/ Respiratory/ Chest pain center/ Emergency, etc.||CRP, PCT, Stroke, Myocardial infarction, Heart failure, Blood glucose, Tumor testing, etc.|
|3||Community clinics||Urine, Hepatitis, Glycohemoglobin testing, etc.|
|4||Self-test and Healthcare management||Ovulation, Pregnancy, Blood pressure, Blood sugar, Blood cholesterol testing, etc.|
|5||Epidemic control||Influenza virus, Coronavirus, Ebola virus, etc.|
|6||Military medicine and Disaster rescue||CRP, Blood gas and electrolyte analysis, Rapid coagulation testing, Malaria, etc.|
|7||On-site administration||Anti-drug, Drunk driving testing, etc.|
|8||Food safety supervision||Pesticide, Veterinary medicine residue testing, etc.|
From the perspective of reading results, POCT can be classified as three types which are qualitative testing, semi-quantitative testing and quantitative testing.
- Qualitative test strip – Reading the testing result directly from the reaction color. Commonly used in pregnancy, drug abuse and HIV test.
- Semi-quantitative test strip – Similar as qualitative testing. Result falls into a value range shown a colorimetric card.
- Quantitative test system – Getting the exact indicator’s value and read the result associated with the pre-defined reference range on the report.
Qualitative test strip
Semi-quantitative test strip
Quantitative test system
POCT Technique Platform
Colloidal Gold-based Immunochromatographic Assay
Also called immune gold labeling technique. It is an immune diagnostic technique based on labeling the second antibody with colloidal gold. Colloidal gold is a sol or colloidal suspension of nanoparticles of gold in a fluid, usually water. The colloid is usually either an intense red color (for spherical particles less than 100 nm) or blue/ purple (for larger spherical particles or nanorods). The following drawing illustrates the test principle.
Immunofluorescence Assay (FIA)
It is also called fluorescent antibody technique. The mechanism is marking a fluorescent dye on the antibody (or antigen) that does not affect the activity of the antigen and antibody. After binding with its corresponding antigen (or antibody), the fluorescent response is presented by illuminating. The instrument determines the analyte concentration by detecting the strength of the light signal. Currently, TRFIA (time-resolve fluorescence immune assay) and QDs (Quantum Dots) assay are more recognized technique in this field.
Chemiluminescence Immunoassay (CLIA)
Chemiluminescence (CL) is defined as the emission of electromagnetic radiation caused by a chemical reaction to produce light. Chemiluminescence immunoassay (CLIA) is an assay that combine chemiluminescence technique with immunochemical reactions. Similar with other labeled immunoassays (RIA, FIA, ELISA), CLIA utilize chemical probes which could generate light emission through chemical reaction to label the antibody.
CLIA have three different label systems according to the difference of physical chemistry mechanism of the light emission.
Label Chemical Directly Involved in the Light Emission Reaction
This kind of chemical with special structure can transfer to an excited state through chemical reaction. Photons would be released when the chemical fell to ground state from the excited state. The typical chemical is acridinium ester and its derivatives. Exposure of an acridinium ester label to an alkaline hydrogen peroxide solution triggers a flash of light. A subsequent development has been the acridinium sulfonamide ester labels. It is also triggered by alkaline hydrogen peroxide to emit a flash of light. The light emission mechanism of acridinium ester is shown in Figure 1.
Figure 1. Mechanism of acridinium ester mediated chemiluminescence.
Acridinium labeled compounds have 100 times stronger chemiluminescence intensity compared to luminol labeled ones, and acridinium esters have the dominant feature that they do not lose the luminescence efficiency even after binding to antigen or antibody. And the chemistry is simple that no enzyme needs to be involved.
Enzyme Catalyzed Light Emission Reaction
This type of chemiluminescence utilizes enzymes to label antibody. Technically speaking, it is an enzyme linked immunoassay that uses luminescent chemical as substrate instead of chromogen. The most widely used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP), each has its own luminescent substrates. Figure 2 shows the detail about HRP-Luminol system (Figure 2 a) and AP-AMPPD system (Figure 2 b). Luminol is a very common chemiluminescent substrate used for detection of HRP. HRP catalyzes the decomposition of luminol in the presence of peroxide to produce an excited state intermediate. Flashes of visible light (maximum at 425nm) is emitted on decay of the singlet intermediate. AMPPD is a derivative of 1, 2-dioxetane substrates. It has a similar mechanism of chemiluminescence. On enzymatic cleavage of the phosphate group, this compound becomes destabilized and decomposes via an intermediate anion, AMPD, which is moderately stable. The wavelength of maximum light emission is 470nm.
Figure 2. Mechanism of Luminol-HRP (a) and AMPPD-AP (b) chemiluminescence system.
Redox Reaction Mediated Light Emission Reaction
Another CL system is noteworthy because the reagent is regenerated and thus can be recycled. This system utilizes ruthenium tris-bipyridine (bpy) as label, involves reaction of Ru(bpy)33+ and Ru(bpy)3+ to produce an excited state of Ru(bpy)32+, a stable species which decays to the ground state by emitting an 620 nm orange emission. Ru(bpy)33+ and Ru(bpy)3+ can be electrogenerated from Ru(bpy)32+ by reduction at approximately -1.3 V, and oxidation at approximately + 1.3 V (Figure 3) . This system is dedicated for electrochemiluminescence with ultrahigh sensitivity and specificity.
Figure 3. Mechanism of Ru(bpy)3 electrochemiluminescence system.
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Last updated: Wednesday, October 19th, 2022 16:18 GMT+8