Development of instruments for fluorescence-based PCR analysis

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Modern PCR analysis systems use fluorescent markers to detect viruses with high specificity and selectivity. The development of customized instruments that use fluorescence for detection is very challenging. The resulting fluorescence signals are often very small and easily influenced by external parameters. Our experts support and advise your customers in the development and production of their fluorescence analysis instruments.

Basic principle of fluorescence. Fluorescent markers excited with light are emitting light of a different wavelength.

Fluorescent markers in PCR analysis

After excitation by light, fluorescent substances spontaneously emit light of a different wavelength than the excitation. In this process, electronic transitions in the molecule are excited. Both the spectrum with which a substance can be excited and the spectrum of the emitted light are characteristic of each fluorescent substance. Their characteristic behavior allows different fluorophores to be present at the same time and still be distinguished from each other. PCR instruments used to detect SARS-COV-2 use different fluorophores simultaneously. For the accurate detection and discrimination of different fluorophores, a careful design of the excitation and detection spectra is necessary.

Coronavirus COVID-19 symbolic design 3d-illustration
The PCR analysis methods used for COVID-19 diagnostics also use fluorescent markers. The polymerase chain reaction is a process for amplifying DNA. In analytical methods of quantitative PCR analysis (qPCR for short), fluorescent dyes are added to the PCR process and become incorporated into the DNA. Thus, the magnitude of the fluorescence signal is a measure of the amount of DNA formed. Three or more different markers are usually used simultaneously to identify the virus DNA. This is necessary to clearly detect the virus. At the same time, the use of multiple fluorescence markers causes the central challenge for the optical system design.
Development of fluorescence detection systems

Development of fluorescence detection systems

For the development of fluorescence-based devices, it is essential to understand the physical and chemical processes to be detected and to study their dependence on environmental fac-tors in detail in the laboratory. Smallest changes in chemistry (e.g. PH) or physic parameters (e.g. temperature) can affect the light output in fluorescence. DIOPTIC develops customized miniaturized prototypes and can comprehensively characterize them in detail in the laboratory.
A great deal of expertise is required for the selection of optical components. Illumination with standard LEDs is cost-efficient but brings new complexities, such as a broad excitation spectrum or large production variations. LEDs in excitation may require the use of additional optical filters.

customized miniaturized prototypes
Our experts can consider these issues already in the concept phase of a product development and simulate them precisely in the optical design. Thus, the entire optical path from illumination, sample illumination, fluorescence to detection can be developed digitally.
The complexity of such an instrument increases when several fluorescent markers are present simultaneously and low detection limits are desired. Our interdisciplinary team consisting of experienced chemists, physicists, optical engineers and hardware developers is well positioned to meet these challenges. Contact us to discuss your fluorescence application. We will find the perfect solution for every customer and every inspection task.

Our experts welcome your inquiries on this topic and will be happy to provide you with advice and assistance.

You will be advised by our expert: Dr. Benjamin Kühnreich ( | Phone: +49 6201 650 40-00