Fluorescent nanoparticle is a general term that many people assume means any nanoscale material that produces fluorescence upon excitation with incident light. However, there is actually a thing called a conjugated polymer nanoparticle (CPN) that is very different from the fluorescent dyes (think Alexa fluorophores) that many of us are used to. These CPNs produce higher intensity fluorescence than dyes (up to 1000x brighter!), are stable and much less susceptible to quenching, and don’t contain toxic cadmium like quantum dots. These conjugated polymer fluorescent nanoparticles will likely be at the forefront of future bioimaging methods.
An enzyme immunoassay is used to detect the presence of a target protein in a biological sample. A more common name for an enzyme immunoassay is the enzyme linked immunosorbent assay (ELISA). These assays are the cornerstone of most established clinical tests, and are typically the gold star diagnostic method. The ELISA is also used in many research environments because of its reliability and sensitivity. It is commonly able to detect protein down to concentrations as low as single picograms per milliliter. The sensitivity depends on the antibody-antigen pair and on the performance of the enzyme-substrate pair. Enzyme immunoassays are labeled assays because they don’t detect a protein binding event (between antibody and antigen) directly. Instead, these assays rely on an enzymatic reaction between an enzyme-conjugated label and a substrate to produce a colorimetric, chemiluminiscent, or fluorescent product. These products are then detected via a spectrophotometer or fluorometer and compared to a standard curve to produce quantitative concentration information about the target protein in the experimental sample.
The enzyme linked immunosorbent assay (ELISA) is a gold standard method for protein and antibody detection. It is routinely used for clinical lab work and is often used in research and development. The assay is based on the lock-and-key specificity between an antibody-antigen pair. Antibodies are naturally made by the adaptive immune system to recognize a specific piece of an antigenic protein. This is what allows the body to identify and fight invading pathogens so effectively. Since antibodies have such a fundamental role in most biosensing and diagnostic systems it is very easy to purchase them or have them custom-made to recognize a protein of interest. The ELISA assay is performed in 96- or 384-well plates that are treated to favorably adsorb antigen or antibody depending on the type of ELISA being performed. The ELISA assay is built up from the bottom of each well; it doesn’t float freely in the well. As a result, the unbound proteins are easily washed away and only the target remains to be quantified.
The concept of an antigen and antibody pair is central to modern medicine and biotechnology. These proteins match like a lock and key, with equisite specificity. The interactions are non-covalent, but have equilibrium constants ranging from 105 to 1012 M-1 Antibodies and antigens are proteins: polypeptide chains of amino acids. The IgG antibody is composed of four polypeptide chains, two heavy and two light, organized into a ‘Y’ shape. The base of the Y is called the Fc region, while the two tips are known as the Fab region.
Chemiluminescent immunoassays (CLIAs) are excellent assays for high-throughput, low analyte concentration and time sensitive testing and isolation. Using coated magnetic beads, such as streptavidin beads, as the reagent in a CLIA is an easy and established technique favored among many clinical scientists.
When scaling up a process using a traditional magnetic separation rack, the percentage of bead and biomolecule losses significantly increases with an increase in volume. One way of dealing with this problem is by applying a higher force at longer distances. But for this to work, you must apply this greater force without increasing the forces in the retention area during the magnetic separation process, in order to avoid irreversible aggregation.
If one wants to scale up production from small lab lots to full-scale large lots, a non-homogenous magnetic separation process will result in lot-to-lot inconsistencies. Homogenous biomagnetic separation conditions, however, guarantee consistent results regardless of production scale.
Due to the inherent properties of classic non-homogenous biomagnetic separators, beads can aggregate during the magnetic separation process. When this happens, technicians try to resolve the magnetic beads separation problem by using special resuspension techniques like the sonication method. But problems with resuspension can ultimately lead to end-product variability, especially if aggregation is not detected early.
In-lot consistency is the key to reproducibility at the level of a kit. Unfortunately, in non-homogenous systems irreversible aggregation is one of the main sources of in-lot variability. If all of the beads are exposed to the same force as they are in homogenous magnetic systems, the risk of aggregation is greatly reduced. Because of this, it is important to know how to avoid irreversible aggregation problems during a magnetic separation process.