What is biochemical analysis?

Biochemical analysis corresponds to detection methods that determine carbohydrates, enzymes, proteins, lipids, inorganic elements, nonprotein nitrogen, liver function, and other characteristics through several biochemical reactions. Biochemical analysis is employed routinely in hospitals, healthcare institutes, and other testing facilities to monitor diseases and disorders. Biochemical assays such as ELISA analysis are employed rapidly in high throughput screening. 

Biochemical screening uses purified target proteins and measures the binding or inhibition activity in vitro. Generally, researchers conduct biochemical assays in a competitive format where the compound of interest displaces a known substrate or ligand. These assays are usually performed in 384-well plates that provide good throughput, screening volume, and experimental cost. The current article discusses the future trends and challenges in the field of biochemical assays. 

Technologies and trends in biochemical assays

Researchers often prefer fluorescent methods over conventional radiolabeled ligand assays because of their higher sensitivity, flexibility, and ease of handling. In total fluorescence intensity assay, the signal intensity from a specific well section is incorporated over a fixed time. These assays use substrates that generate fluorescent signals after enzymatic reaction. 

On the other hand, fluorescent resonance energy transfer has an acceptor that absorbs the energy of a fluorescent donor through dipole-dipole interactions. However, the transfer efficiency is based on spectral overlap between the acceptor and donor, distance, and relative orientation. Another variation of fluorescent resonance energy transfer, time-resolved fluorescence resonance energy transfer, uses lanthanide complexes consisting of long-lived fluorescence as acceptors. The acceptor-emitted fluorescence is then gated for decaying the emission of shorter-lived fluorophores. Critically, the difference in the large stroke shift of acceptor emission and donor excitation minimizes the background noise. Lanthanide fluorescence emission comes in multiple narrow wavelengths compared to a broad emission of organic dyes. This characteristic allows multiplexing using fluorescent acceptors. Different systems available commercially employ diverse donors such as europium chelates, terbium and europium chelates, and europium and terbium cryptates. 

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A less common approach in high throughput screening is fluorescence lifetime analysis. This approach employs time-bound data acquisition to identify the lifespan of fluorescent species in the sample. Ideal dyes for fluorescence lifetime analysis are ruthenium complexes with a lifespan of around 20 ns. Lifetime measurements of fluorescence species are less sensitive to the color of other test compounds. Besides, two lifespans are fit when evaluating the data. This approach removes short-lived elements similar to time-resolved fluorescence resonance energy transfer. 

The emission of fluorescence occurs at a longer wavelength compared to the wavelength of excitation light. In contrast, Alphascreen, a known radioactive assay, employs singlet oxygen sensitization to generate shorter wavelength emission. This shorter wavelength avoids fluorescence artifacts. This assay method conjugates the molecule of interest on two different beads. The donor bead has photosensitizer when stimulated, produces singulate oxygen. Once the donor and acceptor beads come in contact, a chemiluminescence reaction emits light at shorter wavelengths. Singlet oxygen molecules can readily diffuse at large distances, facilitating the measurements of binding events across large binding partners.