As a core analytical tool in modern laboratories, the multifunctional enzyme-linked immunosorbent assay (ELISA) integrates multiple optical detection technologies to achieve precise quantitative and qualitative analysis of biomolecules, cell activity, and chemical substances. The core technical principle is based on Lambert Beer's law and fluorescence resonance energy transfer mechanism, combined with high-precision optical path design and multi-mode detection module, to construct a full scene detection system covering UV visible absorption, fluorescence, chemiluminescence, and time-resolved fluorescence.
Core technical principles
Light absorption detection: using xenon lamp or LED light source, after being separated by a monochromator, a specific wavelength monochromatic light is formed, which penetrates the sample in the microporous plate. By detecting the transmitted light intensity through a photodiode and combining it with Lambert Beer's law (A=ε cl) to calculate the sample concentration, it is suitable for quantitative analysis of nucleic acids and proteins.
Fluorescence detection: Using excitation grating spectroscopy technology, fluorescent markers (such as FITC, GFP) are excited by specific wavelength light to detect their emitted fluorescence intensity. The core innovation lies in the design of the four grating optical path, which supports independent adjustment of excitation/emission wavelengths, with a minimum Stoke shift of 18nm, and can effectively separate background noise.
Chemiluminescence detection: based on the light signal generated by enzymatic or chemical reactions, without the need for external excitation light sources. Detecting glow/flash type luminescence reactions through dark current photon counting level photomultiplier tubes (PMT), with a dynamic range of ≥ 7 orders of magnitude, suitable for ultra trace analysis scenarios such as ATP detection and dual reporter gene analysis.
Innovation in detection mode
Time resolved fluorescence (TRF): Using lanthanide elements (such as europium and terbium) as markers, utilizing their millisecond level fluorescence lifetime characteristics, background interference is eliminated through time delay detection, and the signal-to-noise ratio is increased by more than 10 times. It is widely used in hormone detection and viral hepatitis biomarker analysis.
Fluorescence Resonance Energy Transfer (FRET): Energy transfer detection is achieved by changing the distance between donor acceptor fluorescent molecules, combined with TRF technology to form TR-FRET mode. After donor excitation, the acceptor emits long wavelength fluorescence, which is suitable for protein interaction and acceptor ligand binding research.
Cell imaging integration: Innovatively integrating oblique illumination light path and CCD imaging technology, using LED light source and laser automatic focusing to achieve label free cell counting and fluorescence labeled cell morphology analysis. Compared to traditional enzyme-linked immunosorbent assay (ELISA) instruments, the data dimension has been expanded to include parameters such as sample area and pore coverage, supporting high content screening needs.
Technological breakthrough direction
The current research and development focus is on dynamic range automatic selection, multi gas control live cell detection modules, and AI driven data analysis algorithms. For example, intelligent optimization of detection parameters can be achieved through SkanIt software, which can automatically switch PMT gain based on the signal strength inside the hole, ensuring that the inter batch detection CV value is less than 1%. In the future, with the integration of quantum dot labeling and surface plasmon resonance technology, multifunctional enzyme-linked immunosorbent assay (ELISA) is expected to achieve single-molecule level detection, providing stronger technical support for precision medicine and synthetic biology.