According to the WHO (World Health Organization), early diagnosis of diseases is one of the key factors in the world to save lives and reduce the cost of treatment. Faced with these recommendations, in vitro diagnostic tests can prevent, detect, diagnose, evolve, monitor and manage a pathology, but also select and follow a therapy. Current in vitro tests are designed biological products coupled with fluorescent molecules to detect biomarkers responsible for diseases. These dyes emit a signal that can be observed and quantified and represent one of the central elements to diagnose a pathology.
Today, the detection of biomarkers is addressed by several products already marketed whose performance is moderate. Specifically, developed in the 2000s, fluorescent markers currently used don’t answer the needs of the market for a much more sensitive detection, accurate, fast and cheaper.
Bright-Dtech™ technology is a HK-Dtech solution to improve the sensitivity of detection of biomarkers. We develop high performance fluorescent nano-molecules with unique characteristics. Thanks to their properties, to their high efficiency and to their simplicity of use, this new technology will replace the fluorescent molecules on the market.
Replace fluorescent markers by nanoparticles with high performance properties.
After 10 years of research, development and collaboration, Bright-Dtech™ makes it possible to find a new solution for your experiments, improve your tests or solve your detection problems. To better understand our technology, The summary of unique features of Bright-Dtech™ is shown in the following figure:
The Bright-Dtech™ Technology – Which difference?
Our technology stands out thanks to its fluorescent properties eliminating the defects of current technologies. Among the products on the markets, 3 categories of fluorescent molecules stand out by the habit of use of the customer. A comparison between these 4 types of molecules and Bright-Dtech™, the technology of HK-Dtech is presented in the table below:
This comparative table differentiates the advantages and the disadvantages of each technology according to the essential properties for the user. For example, brightness is an essential parameter of comparison. Strong brightness implies more accurate detection and reduce detection limits. As a result, this new technology detects new unexploited biomarkers for the identification of pathologies, for early disease diagnosis or to improve measuring devices’ performance.
- Immense brightness: 100-1000x brighter than Quantum dots.
- Stable dispersion in solution: Nano-colloidal system.
- Non-toxic: Heavy-metal free, organic.
- Several colors: Red, yellow, green & blue, as well as infrared.
- Familiar surface ligand chemistry: For attaching targeting biomolecules (Ab, oligonucleotides, streptavidin, etc.).
- Storage at ambient temperatures: Unless linked to a biological molecule, then store at 4°C.
Another point related to brightness is the reduction of biological materials consumed that induces cost reduction for detection kits. In addition, the combination with the specificity and the lifetime increases the properties mentioned above compared to the products currently used. The modulation and the control of the surface of the nanoparticle is a parameter not mentioned in the table but allows to Bright-Dtech™ to distinguish itself from other technologies and brings a significant added value because it is important to know the number of biological entities that are added around the nanometric particle.
The Bright-Dtech™ Technology – What is the secret of Bright-Dtech™?
Bright-Dtech™ uses the technology of lanthanides.
The first lanthanide was discovered in Ytterby, a village in Sweden by Lieutenant Carl Axel Arrhenius in 1787. In the form of black mineral called gadolinite, it is Pr. Gadolin who have isolated the yttrium oxide. In 19th century, other lanthanides have been discovered and the exact number of lanthanides have been known from the use of X-ray crystallography. “Rare earth” is named due to the difficulty in separating lanthanides from minerals and not of their abundance because Earth’s crust contains a large quantity.
Lanthanides are the chemical elements having the atomic number Z from 57 (lanthanum) to 71 (lutetium). They are the elements of f block and with yttrium and scandium, they are regarded as part of the « rare earths ». Their general electronic configuration is [Xe] 4fn5d16s2 where n is included between 0 to 14 and represents the number of electrons and [Xe] corresponds to the electronic configuration of the noble gas xenon. In aqueous solvents, lanthanide ions are under the state Ln3+ where they are the most stable in most cases.
Each lanthanides elements have different optical and magnetic properties. For example, although most lanthanides ions are luminescent except La3+ and Lu3+ which have empty or full 4f orbitals. In term of magnetic properties, Ga3+ is usually used as an MRI contrast agent as it is a paramagnetic material with a high spin and high relaxitivity.
Today, lanthanides are mainly used as catalysts, magnets (superconductors), and present in batteries and in optical lasers and fibers but their specific properties have attracted in life science and in the medical areas.
The power of the nanoparticle resides in its nanometric structure. Nanoparticles are small object between 1 to 100 nm and are used in many sectors: healthcare (Markers, biomaterials), environment preservation (flexible solar cells), cosmetic industry (sunscreen) for example. They have many advantages, like small size, large surface area for functionalization, good stability.
The “nanoparticles + lanthanides” combination is the key to the advantages of HK-Dtech.
The Bright-Dtech™ Technology – Why Bright-Dtech™ is so bright?
With a large energy gap between the lowest excited state and the highest ground level, each lanthanide has a specific spectroscopic signature and it is easy to distinguish between them but also other fluorophores or transition metal complexes. The spectral range of lanthanides is large and they emit a strong luminescence either in the visible region (Sm3+, Eu3+, Tb3+, Dy3+) or in the near infrared region (Nd3+, Er3+, Ho3+, Tm3+, Yb3+).
- A long emission lifetime, that can reach some milliseconds for Terbium and Europium.
- With a large pseudo stokes shift, the excitation and emission wavelengths are distinguishable.
- With the 4f orbitals located close to the nucleus and protected from outside by orbitals 5s and 6p, emission bands are very narrow and independent of the surrounding environment.
The problem of lanthanides is the difficulty to absorb the light from excitation source according to the Laporte selection which forbids the f-f transition and leads to very weak molar extinction coefficients. HK-Dtech’s knowledge and know-how lies in the design of nano-markers that solve these difficulties and take advantage of the incredible advantages of lanthanides.
After many years of research, HK-Dtech has developed an ultrabright product combining all the advantages of lanthanides and of nanoparticles.
To understand the brightness of our marker, we need to understand some indications about the light:
- Molar absorption coefficient
- Quantum yield
Molar absorption coefficient
The molar absorption coefficient is the capacity of a molecule to absorb at a specific wavelength. From the Beer-Lambert law and with the knowledge of the concentration of the sample, it is possible to determine molar absorption coefficients for each wavelength and to know the nature of energy levels and electronic transitions with absorption bands by absorption spectroscopy.
- I0: Incident light intensity
- I: Transmitted intensity
- l: Optical path length (cm)
- c: Concentration of sample (mol. L-1)
- ε: Molar absorption extinction (L. mol-1cm-1)
- A: Absorbance
These data are important for the study of emission properties and quantum yield.
The luminescent quantum yield for a chemical species is the number of emitted photons per number of absorbed photons.
The quantum yield is often determined by a comparison method with a known quantum yield. This method is to compare the emission spectrums and absorption bands. The refractive index of the medium (n) and the intensity of the incident beam (I°) are used in the calculation to get the quantum yield.
It is possible to simplify this equation by using the same conditions and same solvents for each sample, if samples are diluted (A <= 0.05) and the spectrometer is for constant excitation intensity I0/Alpha = constant whatever the wavelength l), the quantum yield can be written as a function of the absorbance (A) and integration of emission intensity (S).
Luminescence quantum yields can also be determined in the solid-state or solution by an absolute method with an integrating sphere. This method described by Rohwer and Martin allows to measure and calculate the quantum yield of solid using the following equation:
With the luminescent quantum yield and the molar absorption coefficient, the brightness can be calculated by the following equation:
A bright marker must absorb light efficiently i.e. to have a high molar absorption coefficient and must emit as many photons as possible i.e. to have a great quantum yield.
The brightness allows the comparison between luminescent systems. In a biological system analyzed by fluorescence microscopy, it is important to have a stronger signal that the autofluorescent background . Various organic fluorescent molecules show a high brightness but some compounds with a bigger size improve this value. It is therefore important to combine brightness and size which is not taken into account in these calculations. Thus, the brightness can reach values up to 106 M-1cm-1 or more with fluorescent proteins such as phycoerythrin and semiconducting luminescent nanoparticles (Quantum Dots).
The Bright-Dtech™ Technology – What is the lifetime (and TRF)?
When a luminescent compound is excited, the excited state is populated and the return to the ground state results in the emission of light. This de-excitation decreases exponentially with time, following the law:
- I0 : Intensity after excitation
- Iem: Measured intensity at time t
- t : Time
- T : Excited-state lifetime
The lifetime corresponds to the time a fluorophore stays in its excite state. Fluorescent molecules have very short luminescent lifetimes, in the order of nanosecond but some systems have lifetimes reaching to millisecond or more.
With the used of fluorophores with a long lifetime like lanthanides, TRF measurement could be done in order to remove the background. The principle of time-gated detection is to implement a delay before acquisition of emission of the sample after a pulsed excitation. Experimentally, a delay of the order of 10 – 100 ns after an excitation from pulsed excitation source is necessary to reduce all background intensities. By using chromophores with a long excited-state lifetime (milliseconds and seconds), this detection allows the elimination of the background of the biological environment, keeps a high intensity of emission of the probe sample and to significantly enhances the signal to noise and signal to background intensity ratios.
The Bright-Dtech™ Technology – What is the advantage to use Bright-Dtech™ in FRET?
Time-resolved fluorescence energy transfer (TR-FRET) is the practical combination of Time-Resolved Fluorescence (TRF) with Förster resonance energy transfer (FRET) that offers a powerful tool for drug discovery researchers. TR-FRET combines the low background aspect of TRF with the homogeneous assay format of FRET. The resulting assay provides an increase in flexibility, reliability and sensitivity in addition to higher throughput and fewer false-positive/false-negative results. FRET involves two fluorophores, a donor and an acceptor. Excitation of the donor by an energy source (e.g. flash lamp or laser) produces an energy transfer to the acceptor if the two are within a given proximity to each other. The acceptor in turn emits light at its characteristic wavelength.
The FRET aspect of the technology is driven by several factors, including spectral overlap and the proximity of the fluorophores involved, wherein energy transfer occurs only when the distance between the donor and the acceptor is small enough. In practice, FRET systems are characterized by the Förster’s radius (R0): the distance between the fluorophores at which FRET efficiency is 50%. For many FRET fluorophore pairs, R0 lies between 20 and 90 Å, depending on the acceptor used and the spatial arrangements of the fluorophores within the assay. Through measurement of this energy transfer, interactions between biomolecules can be assessed by coupling each partner with a fluorescent label and detecting the level of energy transfer. Acceptor emission as a measure of energy transfer can be detected without needing to separate bound from unbound assay components (e.g. a filtration or wash step) resulting in reduced assay time and cost.
Homogeneous, mix-and-read TR-FRET assays offer advantages over other biomolecular screening assays, such as fluorescence polarization (FP) or TRF assays. In FP assays, background fluorescence due to library compounds is normally depolarized and background signal due to scattered light (e.g. precipitated compounds) is normally polarized. Depending on the assay configuration, either case can lead to a false-positive or false-negative result. However, because the donor species used in a TR-FRET assay has a fluorescent lifetime that is many orders of magnitude longer than background fluorescence or scattered light, the emission signal resulting from energy transfer can be measured after any interfering signal has completely decayed. TR-FRET assays can also be formatted to use limiting bioreceptor and excess tracer concentrations (unlike FP assays), which can provide further cost savings. In the case of TRF assays, a wash step is required to remove unbound fluorescent reagents before measuring the activity signal of the assay. This increases reagent use, time to complete the assay, and limits the ability to miniaturize the system (e.g. converting from a 384-well microtiter plate to a 1536-well plate). TR-FRET assays take advantage of the required proximity of the donor and acceptor species for the generation of the signal without a washing step.
Additionally, this method is preferred by some researchers as it does not rely on radioactive materials to generate the signal to be detected. This avoids both the hazards of using the materials and the cost and logistics of storage, use, and disposal.
The Bright-Dtech™ Technology – Why is so easy to couple Bright-Dtech™?
The coupling between biological entities and nanoparticles is the key for optimal detection. This step is essential to avoid background noise but also to avoid losing often expensive product.
At HK-Dtech, this connection has been worked on to have a strong, and stable coupling that does not disturb the desired interactions. In addition, one of the strong points of our technology is the ability to modify the surface chemistry of the nanoparticle to allow optimal, controlled, and customizable use of the nanoparticles.
Thus, we have developed, Link-Dtech™, a coupling kit with “Ready to couple” nanoparticles capable of being linked to biological entities without complex coupling chemistry. In parallel, we also offer, on request, coupling kits with specific coupling functions (Carboxyl, Amine, isocyanate, N3, etc.) and services to couple specific biological materials with our Bright-Dtech™ nanoparticles.
To prepare antibodies free of amines and phosphates, other proteins and preservatives that can interfere with the conjugation process. At HK-Dtech, we mainly use Zeba™ Spin Desalting Columns, 7K MWCO, 0.5 mL and Amicons from Merck.
We also offer you a customizable coupling service in our laboratory by our experts in bioconjugation, chemistry and biology.