형광면역분석(법)/fluorescence immunoassay

일종의형광반응으로 Sample 형광 물질이 특정 파장의 빛을 흡수하면 형광 물질의 분자가 여기(excitation)되었다가, 다시 원래의 상태로 돌아오면서 흡수한 빛과는 다른 파장(emissino)의 빛을 내는 반응.

* 레이저, LED 등에 적용되는 원리와 유사

Fluorescence Fundamentals

출처 : https://www.thermofisher.com/kr/ko/home/references/molecular-probes-the-handbook/introduction-to-fluorescence-techniques.html

Fluorescence quantum yields (QY) and lifetimes (τ) for Alexa Fluor dyes.

https://www.thermofisher.com/kr/ko/home/references/molecular-probes-the-handbook/tables/fluorescence-quantum-yields-and-lifetimes-for-alexa-fluor-dyes.html

https://www.thermofisher.com/kr/ko/home/references/molecular-probes-the-handbook/introduction-to-fluorescence-techniques.html

A guide to how fluorescence and tandem dyes work.

 출처 : https://www.abcam.com/secondary-antibodies/fluorescence-guide

 

How does fluorescence work?

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1. Electromagnetic energy from a laser set at the correct wavelength will provide the right amount of energy to an electron in the fluorescent dye molecule. This is the signature excitation wavelength for the molecule. The energy is absorbed by this electron.
2. On absorption of this energy, the electron moves to an excitation state at the next energy level (Es). 
3. Finally, this energy is released in the form of a photon (fluorescence) and the electron moves back down to the lower energy level. The amount of energy released will be determined by how far the electron drops down the energy levels which will always be the same in the same fluorescent molecule. This will determine the wavelength of the photon, and the color of the fluorescence observed giving the fluorescent dye its signature emission wavelength.

​How do tandem dyes work?

Tandem fluorescent dyes are conjugated 'dual' fluorescent molecules, for example, PE-Cy5. On the antibody, they will be close enough so that the energy can be transferred between the two. The laser excitation wavelength used will excite the donor molecule only (eg PE) – it will not be the correct wavelength to excite the acceptor molecule. The energy then released from the donor molecule will be at the correct wavelength to excite the electron in the acceptor molecule. The acceptor molecule will then release energy in the form of a photon at its signature wavelength.

So, for example, PE-Cy5 will excite at the excitation wavelength for PE (565 nm) and emit at the emission wavelength for Cy5 (670 nm).

1. Electromagnetic energy from a laser set at the correct wavelength will provide the right amount of energy to an electron in the donor fluorescent dye molecule. This is the signature excitation wavelength for the molecule. The energy is absorbed by this electron.
2. On absorption of this energy, the electron moves to an excitation state at the next energy level (Es).
3. Energy is released from this electron in the form of a photon. The electron moves back down to the lower energy level. The energy is released in the form of a photon. This then excites an electron in the tandem dye molecule which moves up to the next energy level.
4. Finally, this energy is released in the form of a photon (fluorescence) and the electron moves back down to the lower energy level. The amount of energy released will be determined by how far the electron drops down the energy levels which will always be the same in the same fluorescent molecule. This will determine the wavelength of the photon, and the color of the fluorescence observed.

time-resolved fluorescence (TRF)

출처 : https://www.abcam.com/content/time-resolved-fluorescence-trf-introduction

What is time-resolved fluorescence?

Time-resolved fluorescence (TRF) is very similar to standard fluorometric detection. The main difference between the two measurements is the timing of the excitation/emission process. During standard fluorometric detection, excitation and emission are simultaneous; the light emitted by the sample is measured while excitation is taking place. In contrast to this, TRF relies on the use of very specific fluorescent molecules, called lanthanide chelate labels, which allow detection of the emitted light to take place after excitation has occurred. The most commonly used lanthanide chelate label is the europium ion (Eu3+).

 

Lanthanides offer several key advantages:

1. A large Stokes shift greatly increases the signal:background (S:B) ratio.
2. A sharp emission peak allows different lanthanides to be easily distinguished from one another and contributes to improved S:B.
3. High fluorescence intensity significantly improves assay sensitivity.
4. A long fluorescence lifetime (µseconds–milliseconds), several orders of magnitude greater than any nonspecific background fluorescence (typically nanoseconds), and stable fluorescent signal enable the fluorescent emission to be read at a time well after any background fluorescence has decayed, delivering a greater dynamic range.

* Stokes shift : 여기되는 물질에서 발광하는 파장이 여기광의 파장보다 길어지는 현상.

Figure 1. Absorbance and emission spectra of europium. Europium has a large Stokes shift, a wide excitation spectrum, and a narrow emission spectrum, typical of lanthanide chelates.