6.3 Quantum dots for biological sensing and imaging
10 min read•august 14, 2024
Quantum dots are revolutionizing biological sensing and imaging. Their unique optical properties, like and , make them ideal for long-term tracking and multiplex imaging. These nanoparticles outshine traditional fluorescent probes in many ways.
Bioconjugation strategies allow quantum dots to be linked to various biomolecules, enhancing their specificity and functionality. However, concerns about toxicity and biocompatibility remain. Researchers are developing strategies to mitigate these issues, paving the way for safer and more effective quantum dot-based biosensors and imaging tools.
Quantum dot biosensing principles
Unique optical properties of quantum dots for biosensing and bioimaging
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Quantum dots possess size-tunable emission, enabling the production of a wide range of colors by varying the quantum dot size
Broad absorption spectra allow for efficient excitation of multiple quantum dot colors using a single light source
Narrow emission spectra of quantum dots result in minimal spectral overlap and improved signal-to-noise ratios compared to traditional fluorescent dyes
High photostability of quantum dots enables long-term imaging and sensing applications without significant photobleaching (fluorescein, rhodamine)
Mechanisms of quantum dot-based biosensing
Quantum dot-based biosensing relies on changes in the optical properties of quantum dots upon interaction with target analytes
Fluorescence quenching occurs when the target analyte reduces the fluorescence intensity of the quantum dot (heavy metal ions, organic pollutants)
Fluorescence enhancement can be observed when the target analyte increases the fluorescence intensity of the quantum dot (certain enzymes, nucleic acids)
Wavelength shift in the emission spectra of quantum dots can be induced by the binding of specific analytes (pH changes, ionic strength variations)
Förster Resonance Energy Transfer (FRET) is a commonly used mechanism in quantum dot-based biosensing
FRET involves the non-radiative energy transfer between a quantum dot (donor) and a fluorescent dye or quencher (acceptor)
The presence of the target analyte modulates the distance or orientation between the quantum dot and the acceptor, affecting the FRET efficiency
FRET-based biosensors can be designed for the detection of various analytes (proteins, nucleic acids, small molecules)
Applications of quantum dots in bioimaging
Quantum dot-based bioimaging utilizes the bright and stable fluorescence of quantum dots for labeling and tracking biomolecules
Quantum dots can be conjugated to antibodies, aptamers, or peptides for specific labeling of proteins, nucleic acids, and cells in vitro and in vivo
The photostability of quantum dots allows for long-term tracking of labeled biomolecules without significant signal loss (single-particle tracking, time-lapse imaging)
Multiplex imaging can be achieved using quantum dots with different emission colors
Simultaneous detection and tracking of multiple biological targets can be performed by exciting the sample with a single light source
Spectral unmixing techniques can be applied to separate the signals from different quantum dot colors (fluorescence microscopy, flow cytometry)
Quantum dots have been used for in vivo imaging of tumors, lymph nodes, and other tissues in animal models (mice, zebrafish)
Bioconjugation strategies for quantum dots
Covalent coupling of biomolecules to quantum dots
Covalent coupling involves the formation of chemical bonds between the biomolecules and the functional groups on the quantum dot surface
Carboxyl groups on the quantum dot surface can be activated using carbodiimide chemistry (EDC, NHS) and reacted with amine groups on the biomolecules (antibodies, peptides)
Amine groups on the quantum dot surface can be conjugated to carboxyl groups on the biomolecules using similar chemistry
Thiol groups on the biomolecules can be directly coupled to the quantum dot surface through the formation of disulfide bonds (cysteine-containing peptides, thiolated DNA)
Factors affecting the efficiency of covalent coupling include the molar ratio of biomolecules to quantum dots, the pH and composition of the reaction buffer, and the reaction time and temperature
Non-covalent bioconjugation strategies
Streptavidin-biotin interaction is a widely used non-covalent bioconjugation strategy for quantum dots
Quantum dots are coated with streptavidin, a tetrameric protein with high affinity for biotin
Biomolecules are labeled with biotin, a small molecule that can be easily incorporated into proteins, nucleic acids, or other targets
The streptavidin-coated quantum dots bind strongly to the biotin-labeled biomolecules, forming a stable and specific bioconjugate
Electrostatic interaction can be employed for the adsorption of positively charged biomolecules onto the negatively charged surface of quantum dots
Positively charged peptides or proteins can be directly adsorbed onto the quantum dot surface without the need for chemical modification
The stability of the electrostatic bioconjugates depends on the pH and ionic strength of the environment and may be affected by changes in these conditions
Other non-covalent bioconjugation strategies include hydrophobic interactions, hydrogen bonding, and metal-affinity coordination (His-tagged proteins)
Factors affecting the performance of quantum dot bioconjugates
The surface chemistry of quantum dots plays a critical role in the efficiency and specificity of bioconjugation
Hydrophilic coatings (PEG, silica) improve the colloidal stability and reduce the non-specific binding of quantum dots to biological components
Functional groups (carboxyl, amine, thiol) provide reactive sites for the attachment of biomolecules
The ratio of biomolecules to quantum dots determines the valency and orientation of the bioconjugates
High ratios may lead to overcrowding and steric hindrance, reducing the accessibility and functionality of the attached biomolecules
Low ratios may result in incomplete surface coverage and decreased signal intensity
The reaction conditions, such as pH, temperature, and buffer composition, affect the kinetics and efficiency of bioconjugation
Optimal pH values depend on the type of functional groups and the stability of the biomolecules (neutral pH for amine coupling, slightly acidic pH for thiol coupling)
Elevated temperatures may accelerate the reaction but may also cause denaturation of the biomolecules
Purification methods, such as size-exclusion chromatography, ultracentrifugation, or dialysis, are necessary to remove the unreacted reagents and to obtain pure and stable quantum dot bioconjugates
Quantum dot toxicity and biocompatibility
Factors influencing the toxicity of quantum dots
The composition of the core and shell materials determines the inherent toxicity of quantum dots
Cadmium and lead-based quantum dots are more toxic than silicon, carbon, or zinc-based ones due to the release of heavy metal ions
Thick and stable shell coatings (ZnS, silica) can mitigate the leakage of toxic ions from the core and reduce the overall toxicity
The surface coating and functionalization of quantum dots affect their interactions with biological systems
Hydrophobic coatings (TOPO, oleic acid) can cause aggregation and non-specific binding to proteins and cell membranes, leading to toxicity
Hydrophilic and biocompatible coatings (PEG, peptides) improve the solubility, stability, and circulation time of quantum dots in biological fluids
The size and shape of quantum dots influence their cellular uptake, biodistribution, and clearance
Smaller quantum dots (<5 nm) can be more easily internalized by cells and may cause more pronounced cytotoxicity than larger ones
Spherical quantum dots are generally less toxic than rod-shaped or irregular-shaped ones due to their lower surface area and reactivity
The route and duration of exposure to quantum dots determine the extent and severity of their toxic effects
Intravenous injection of quantum dots may lead to systemic toxicity and accumulation in organs (liver, spleen, kidneys)
Oral or dermal exposure to quantum dots may cause local inflammation and irritation but may be less harmful than systemic exposure
Strategies to mitigate the toxicity of quantum dots
Using less toxic core materials, such as silicon, carbon, or zinc-based quantum dots, instead of cadmium or lead-based ones
Silicon quantum dots have low toxicity and good biocompatibility due to their biodegradability and low heavy metal content
Carbon quantum dots can be synthesized from natural precursors (citric acid, amino acids) and exhibit excellent water solubility and low cytotoxicity
Applying thick and stable shell coatings, such as ZnS or silica, to prevent the leakage of heavy metal ions from the core
ZnS shell coating can passivate the surface of CdSe or CdTe quantum dots and reduce their cytotoxicity by several orders of magnitude
Silica shell coating provides a biocompatible and chemically inert surface for quantum dots and can be easily functionalized with various biomolecules
Functionalizing the quantum dot surface with biocompatible polymers, such as polyethylene glycol (PEG), to improve their solubility, stability, and circulation time in biological fluids
PEGylation of quantum dots reduces their non-specific interactions with proteins and cells and prolongs their blood circulation time
PEG coating also minimizes the adsorption of opsonins and prevents the rapid clearance of quantum dots by the reticuloendothelial system
Evaluation of quantum dot toxicity and biocompatibility
In vitro cytotoxicity assays, such as MTT or alamarBlue, can be used to evaluate the short-term toxicity of quantum dots on cultured cells
MTT assay measures the metabolic activity of cells based on the reduction of a tetrazolium dye and can indicate the viability of cells exposed to quantum dots
AlamarBlue assay assesses the oxidation-reduction potential of cells and can provide a quantitative measure of the cytotoxicity of quantum dots
In vivo animal studies are necessary to assess the long-term biodistribution, clearance, and toxicity of quantum dots in living organisms
can be used to track the distribution and accumulation of quantum dots in different organs and tissues (liver, spleen, kidneys, lungs)
Blood and urine analysis can provide information on the clearance and excretion of quantum dots from the body
Histological examination of organs can reveal any pathological changes or inflammatory responses induced by quantum dots
The biocompatibility of quantum dots can be enhanced by minimizing their non-specific interactions with biological components and by promoting their specific targeting and uptake by the desired tissues or organs
Functionalization of quantum dots with targeting ligands (antibodies, peptides, aptamers) can improve their specificity and reduce their off-target effects
Optimization of the size, shape, and surface chemistry of quantum dots can enhance their cellular uptake and intracellular trafficking while minimizing their cytotoxicity
Quantum dots vs other fluorescent probes
Advantages of quantum dots over traditional fluorescent probes
Broad absorption spectra and narrow emission spectra of quantum dots
Quantum dots can be efficiently excited by a wide range of wavelengths, allowing for the use of a single excitation source for multiple colors
The narrow emission spectra of quantum dots enable higher signal-to-noise ratios and minimal spectral overlap, facilitating multiplex sensing and imaging
High photostability and resistance to photobleaching of quantum dots
Quantum dots can withstand prolonged exposure to light without significant loss of fluorescence intensity, enabling longer observation times and repeated imaging
Traditional organic dyes (fluorescein, rhodamine) and fluorescent proteins (GFP) are more susceptible to photobleaching, limiting their use in long-term imaging applications
Size-tunable emission color of quantum dots
The emission wavelength of quantum dots can be precisely controlled by varying their size and composition, covering a wide range of colors from the UV to the near-infrared
The ability to tune the emission color of quantum dots facilitates the design of multiplexed assays and the optimization of the signal-to-noise ratio for specific applications
Limitations of quantum dots compared to other fluorescent probes
Large size of quantum dots compared to organic dyes and fluorescent proteins
Quantum dots are typically 2-10 nm in diameter, which is larger than most organic dyes (<1 nm) and fluorescent proteins (2-4 nm)
The large size of quantum dots may hinder their penetration into cells or tissues and may interfere with the function of the labeled biomolecules (steric hindrance, altered binding affinity)
Blinking behavior of individual quantum dots
Quantum dots exhibit intermittent fluorescence emission, known as blinking, which can cause signal fluctuations and complicate single-molecule tracking experiments
The blinking of quantum dots can be reduced by using special surface coatings (thick shells, alloyed cores) or by employing anti-blinking strategies (redox cycling, electron transfer)
Toxicity and long-term fate of quantum dots in biological systems
The potential toxicity of quantum dots, particularly those containing heavy metals (cadmium, lead), remains a concern for in vivo applications
The biodegradation and clearance of quantum dots from the body may be slow and incomplete, leading to long-term accumulation and adverse effects
Strategies to mitigate the toxicity of quantum dots, such as using less toxic materials or applying biocompatible coatings, are being actively investigated
Comparison with other emerging fluorescent probes
Organic dyes, such as fluorescein and rhodamine, are small molecules with well-established bioconjugation chemistry
Organic dyes are widely available and have been extensively used in biological labeling and sensing applications
However, organic dyes suffer from rapid photobleaching, limited brightness, and spectral overlap, which restrict their use in long-term and multiplexed imaging
Fluorescent proteins, such as green fluorescent protein (GFP) and its variants, can be genetically encoded and expressed in living cells
Fluorescent proteins provide a non-invasive and highly specific labeling method for studying protein localization and dynamics in vivo
However, fluorescent proteins have lower brightness and photostability compared to quantum dots and may interfere with the function of the tagged proteins
Upconversion nanoparticles and carbon dots are emerging fluorescent probes with unique properties
Upconversion nanoparticles can absorb multiple low-energy photons (near-infrared) and emit high-energy photons (visible), enabling deep tissue imaging and reduced autofluorescence background
Carbon dots are small (<10 nm), biocompatible, and photostable fluorescent nanoparticles that can be synthesized from renewable sources and functionalized with various surface groups
However, the bioconjugation chemistry and sensing mechanisms of these emerging probes are less developed compared to quantum dots and require further optimization for specific applications