Quantum dots and prion proteins

Abstract

A diagnostics of infectious diseases can be done by the immunologic methods or by the amplification of nucleic acid specific to contagious agent using polymerase chain reaction. However, in transmissible spongiform encephalopathies, the infectious agent, prion protein (PrP^Sc), has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrP^Sc detection is that the pathological form of prion protein is abundant only at late stages of the disease in a brain. Therefore, the diagnostics of prion protein caused diseases represent a sort of challenges as that hosts can incubate infectious prion proteins for many months or even years. Therefore, new in vivo assays for detection of prion proteins and for diagnosis of their relation to neurodegenerative diseases are summarized. Their applicability and future prospects in this field are discussed with particular aim at using quantum dots as fluorescent labels.

Keywords: :

• imaging
• label
• neurodegenerative disease
• prion protein
• quantum dots

Introduction

Alzheimer disease (AD) is characterized by the formation of senile plaques composed of the amyloid-β (Aβ) peptide and neurofibrillary tangles composed of hyperphosphorylated Tau. However, it is the accumulation of Aβ in the brain that appears to be critical for the pathogenesis of AD. The prion protein (PrP, Fig. 1) is involved in neurodegeneration via its conversion from the normal cellular form, PrP^C, to the infectious form, PrP^Sc, which is the causative agent of the transmissible spongiform encephalopathies (TSEs), including Creutzfeldt-Jakob disease (CJD).^2-4 While there is an established role for PrP^C in TSEs, the physiological role of PrP^C has still not been fully elucidated. Roles in metal homeostasis, neuroprotective signaling, lymphocyte activation, neurite growth, synaptogenesis, cellular signaling, cell viability, and cellular response to oxidative stress have all been proposed.^5-10 There are many neuropathological similarities and genetic links between AD and prion diseases. The coexistence of AD pathology in CJD has been reported¹¹ and PrP^C has been shown to co-localize with Aβ in plaques. These PrP^C-Aβ plaques have been found in most CJD patients with associated AD-type pathology and it has been proposed that PrP^C may promote Aβ plaque formation.^12,13 A genetic correlation between PrP^C and AD has also been reported.^14,15 A systematic meta-analysis of AD genetic association studies revealed that the gene encoding PrP^C (PRNP) is a potential AD susceptibility gene and the Met/Val 129 polymorphism in PRNP has been determined as a risk factor for early-onset AD.¹⁶

Figure 1. Three-dimensional (3D) structure of human PrP. The structure of the α-helical form of rPrP(90–231) resembles that of PrP^C. rPrP(90–231) is viewed from the interface where PrP^Sc is thought to bind to PrP^C. The color scheme is as follows: α-helices A (residues 144–157), B (172–193), and C (200–227) in pink; disulphide between Cys-179 and Cys-214 in yellow; conserved hydrophobic region composed of residues 113–126 in red; loops in gray; residues 129–134 in green encompassing strand S1 and residues 159–165 in blue encompassing strand S2; the arrows span residues 129–131 and 161–163, as these show a closer resemblance to β-sheet (155).¹ The data source was the internet proteomic database Expasy (www.expasy.org). For data processing software Matlab version 7.9.0 (The MathWorks, Inc.) was used.

Current Diagnostics of Prion Protein Related Diseases

Diagnostics of infectious diseases is a relatively well-established area. It can be done either by the immunologic methods or by the amplification of a nucleic acid specific to infectious agent using polymerase chain reaction (PCR).¹⁷ However, in TSEs, the infectious agent, PrP, has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrP^Sc detection is that the pathological form of PrP is abundant only at late stages of the disease in a brain. Therefore, the diagnostics of PrP caused diseases represent a sort of challenges¹⁸ as that hosts can incubate infectious prion proteins for many months or even years. During this period, they exhibit no overt clinical symptoms. Incubation period for some human prion diseases can be as long as 40 years.¹⁹ The incubation time of the first human transmissible spongiform encephalopathy “Kuru” described by Prof. Gajdusek in the late 1950s was estimated within the range from 21 to 40 years.^20,21

Immunological detection of PrP^Sc post mortem

Generally, the majority of analytical diagnostic methods rely on the proteolytic removal of endogenous PrP^C prior to detection of PrP^Sc (Fig. 1). PrP^Sc is relatively resistant toward proteolytic degradation whereas PrP^C is entirely digested by Proteinase K. Identical treatment leads to removal of a variable number of N-terminal amino acids in the case of PrP^Sc. This results in the appearance of three distinct bands, corresponding to di-, mono-, and un-glycosylated forms of PrP, upon Western Blotting. Several techniques have been developed to detect PrP^Sc in brain tissues, including Western Blot or other immunoblot methods.^22-24 Quantitative Western Blot analysis revealed the highest expression of PrP^C in cerebellum, obex, and spinal cord. Intermediate levels were detected in thymus, intestine, nervous, heart, and spleen; lower levels were found in lung, muscle, kidney, lymph node, skin, pancreas, and liver.²⁵ Western Blotting coupled with gel electrophoresis is one of the immunodetection methods successfully used for detection of PrP^Sc in tissue extracts.^26,27 After denaturation of the tissue extract by heating with sodium dodecyl sulfate, the sample is analyzed by PAGE and the denatured protein is transferred to a solid support and detected with an enzyme-labeled antibody, often of goat, rabbit and mouse. The specificity of Western Blotting is based on the fact that proteolysis with proteinase K characteristically alters the molecular mass (app. five kDa) of the PrP^c, due to the partial degradation of the N-terminal part of the protein.²⁸ Immunohistochemical analysis detected intense cellular-specific PrP^C staining in neurons, thymocytes and lymphocytes. PrP^C was also detected in the enteric wall, pancreatic islets of Langerhans, myocardium, pulmonary alveolar sacs, renal glomeruli, and dermal epithelial cells.²⁵

Another useful approach is the in situ detection of PrP by immunohistochemistry (IHC). In most IHC procedures, the brain tissue sections are treated to destroy PrP^C with formic acid rather than with protease, since formic acid also enhances the PrP^Sc immunoreactivity.²⁹ A suitable IHC procedure was developed using brain tissue from hamsters that had been inoculated with the transmissible mink encephalopathy agent. Tissue samples were fixed in PLP (periodate, lysine, paraformaldehyde) that contained paraformaldehyde at a concentration of 0.125%. Before the application of IHC technique, tissue sections were deparaffinized and treated with formic acid simultaneously to enhance PrP^Sc immunoreactivity and to degrade PrP^C. Primary antibody was obtained from a rabbit immunized to PrP^Sc extracted from brains of mice with experimentally induced scrapie. Brains from 21 sheeps with histopathologically confirmed scrapie were examined by IHC. In all of these brains, PrP^Sc was widely distributed throughout the entire brain.

In the case of human diseases, diagnosis is based almost exclusively on clinical examination. The disease is then considered as probable depending on the extent to which the clinical symptoms fit the standard guidelines. However, in the case of animals, detection of the only disease-specific analyte, PrP^Sc, can be commercially done.³⁰ From the clinical point of view, the most sensitive and specific method for diagnosing TSE is unquestionably experimental infection of laboratory animals. The animal is injected with a homogenate prepared from the suspicious tissue and the appearance of clinical signs is followed. The disease progress is then confirmed after dissection using classic techniques, such as histology, immunohistology, or Western Blot.³¹ The animal infection is too laborious and time-consuming to be used for routine high-throughput screening.²⁸ Recently, new post-mortem tests have been introduced enabling rapid screening of the suspicious samples. Currently, 5 commercial tests are approved by the European Commission for BSE detection (Prionics-Check Western test, Enfer test, CEA/Biorad test, Prionics-Check LIA test, and Conformational-dependent immunoassay). All these tests are based on the immunodetection of the pathological PrP^Sc isoform, whereas four of them use proteolysis to distinguish PrP^C from misfolded PrP^{Sc 17}. It has to be noted that none of these tests is able to identify infected animal at the pre-symptomatic stage and therefore the risk of the infectious agents entering the food chain is not completely eliminated.

Immunological detection of PrP^Sc in peripheral tissues

The infectivity studies have shown that prion proteins occurred in low amounts in the peripheral tissues, such as lymphoid organs and blood,³² already at early stages of the disease, e.g. during the pre-symptomatic period. Some commercial assays (TeSeETM CJD ELISA and TeSeETM, Table 1) are available to detect PrP^Sc in cerebral and lymphoid tissues of TSE patients. These two assays have been compared by Ugnon-Cafe et al.⁵⁰ using samples from 54 vCJD affected patients and 51 controls. Authors concluded that these tools were rapid and robust for routine in vitro human TSE diagnosis and characterization. CJD could also be diagnosed during the patient’s lifetime by detection of PrP^Sc in the tonsil. A pilot study was undertaken to look at the feasibility of testing for vCJD in deceased donors using tonsillar tissue. Obtaining tonsillar tissue in the immediate post-mortem period was limited by the presence of rigor mortis. Tonsillar tissue was suitable for routine analysis for the occurrence of prion protein associated with vCJD in the deceased tissue donors. In spite of the fact that palatine and lingual tonsil tissues could be obtained in pairs, it was possible, in the majority of cases, to set aside an intact sample for confirmatory testing if required.⁵¹ A method using Western Blot assay with precipitation of streptomycin sulfate has been also reported improving the PrP^Sc detection sensitivity and providing the potential for specific, rapid and flexible determination of low PrP^Sc levels in the specimens not only from the central nervous system, but also from peripheral organs or fluids.⁵²

Table 1. Summary of Limits of Detection (LODs) for prion proteins measured by various methods

Method	LOD*	Ref
Long range resonance energy transfer (LrRET)	0.309 µg/mL**	33
Real-time quaking-induced conversion assay (RT-QuIC)	1 ng/mL (1 fg)	34,35
Mass spectrometry (MS)	15 ng/mL (100 amol)	36,37
Capillary electrophoresis based immunoassay	6 ng/mL**	38
Capillary electrophoresis-based competitive immunoassay	80 ng/mL**	39
Surround optical fiber immunoassay (SOFIA)	10 pg/mL (10 ag)	40
Immuno-quantitative real-time PCR	2.32 × 102 prion epitopes***	41
Time resolved fluorescence immunoassay	50 ng/mL (10 pg)	42
Simulated sandwich immunoassay	10 fg/mL**	43
Amyloid seeding assay (ASA)	1 ng/mL (1 fg)	44
Aggregation-specific enzyme-linked immunosorbent assay (AS-ELISA)	20 ng/mL	45
Aggregation-specific fluorescent amplification catalyzed by T7 RNA polymerase technique (AS-FACTT)	10 pg/mL	45
Flow cytometry	300 µg/mL (10nM)	46
Aptamer-mediated turn-on fluorescence assay	0.3 µg/mL	47
Nanoparticle-based surface-modified fluorescence assay	60 µg/mL (2 nM)	48
Resonance light scattering (RLS)	2 µg/mL (7 × 10^−11 M)	49
Chronopotentiometric stripping analysis	500 µg/mL (25 pmol/5 µL)	5

*Limits of detection were re-calculated on the similar units (grams per mL), the original data are in brackets. **The original data were expressed in gram per mL. ***These cannot be re-caluclated on grams per mL.

A new in vitro amplification technology, designated “real-time quaking-induced conversion (RT-QUIC),” has been described for the detection of tabnormal PrP (PrP^Sc) form in easily accessible specimens, such as cerebrospinal fluid. RT-QUIC method can be applied to other prion diseases, including scrapie, chronic wasting disease (CWD) and bovine spongiform encephalopathy (BSE), and is able to quantify PrP seeding activity when combined with an end-point dilution of samples.³⁴ Solid-state matrix can be used for capturing and concentrating disease-associated PrP. Coupling of this method with direct immunodetection of surface-bound material enabled to distinguish 10⁻¹⁰ dilution of exogenous vCJD prion-protein-infected brain from 10⁻⁶ dilution of normal brain (mean chemiluminescent signal, 1.3 × 10⁵ for vCJD vs. 9.9 × 10⁴ for normal brain; showing an assay sensitivity for vCJD of 71.4% and a specificity of 100%).⁵³

Non-immunological detection of PrP^Sc

In order to avoid the use of antibodies as mentioned in previous paragraph, several spectroscopic methods, such as multispectral UV fluoroscopy,⁵⁴ fluorescence correlation spectroscopy,^55,56 magnetic resonance spectroscopy^57-59 or Fourier-transformed infrared spectroscopy (FTIR)^60,61 have been employed. The main disadvantages of these methods include requirements of expensive and sophisticated equipment as well as skilled operator. Recently, Raman scattering spectroscopy using gold nanorods 3D-supercrystals was used for PrP detection.⁶² Last but not least, sensitive mass spectrometry based method of the PrP quantification in a variety of mammalian species has been presented.³⁶

On the other hand, protein misfolding cyclic amplification (PMCA) has a great potential and it is certainly the most promising approach from the viewpoint of a blood test development. It mimics pathological processes and is similar to the polymerase chain reaction; PrP^Sc is incubated in the presence of PrP^C excess to initiate conversion to PrP^Sc aggregates which are subsequently dispersed by sonication to encourage the formation of new aggregates. The quantity of formed PrP^Sc depends on the number of expansion/sonication cycles performed.^63-66 Detection limits are summarized in Table 1.

In vivo diagnostics

Neurodegenerative diseases can be definitively confirmed by post mortem histopathologic examination of senile plaques (SPs) and neurofibrillary tangles (NFTs) in the brain using stains and dyes that identify these microscopic structures. Advances in in vivo detection of β-amyloid formation and aggregation may further facilitate the drug development for the disease by providing critical information on plaque burden in the living brain. Currently, development of specific molecular imaging agents for direct mapping of Aβ aggregates in the living brain is a research topic being actively investigated. Several research groups have launched programs to successfully identify the biomarkers for imaging Aβ plaques in the brain.^67-69

Even though computer tomography (CT) is not sensitive or specific in the evaluation of the disease, it may show atrophy at the advanced stage of the disease. Magnetic resonance imaging (MRI) is far more sensitive, especially with the addition of fluid attenuation inversion recovery (FLAIR) and diffusion weighted imaging (DWI). MRI of patient’s brain with prion diseases may show mild to moderate generalized atrophy at the early stage of disease and dramatic diffuse atrophy after a relatively short duration of disease; however, this finding is not consistent.⁷⁰

The detection of individual SPs and NFTs in vivo by positron-emission tomography (PET) or single-photon emission CT (SPECT) should improve diagnosis and also accelerate discovery of effective therapeutic agents. About a decade ago, the first successful Aβ plaque-specific PET imaging study was conducted in a living human subject clinically diagnosed with probable AD using the ¹¹C-labeled radiopharmaceutical Pittsburgh Compound B (PiB; [¹¹C]-2-4′-[methylaminophenyl]-6-hydroxybenzothiazole).⁷¹ Since then many PET/SPECT imaging probes have been suggested, e.g. (¹⁸F)-2-(1-[2-{N-(2-fluoroethyl)-N-methylamino} naphthalene-6-yl]ethylidene)malononitrile (FDDNP), (¹¹C)4-N-methylamino-4′-hydroxystilbene (SB-13),⁷² (¹¹C)2-(2-[2-dimethylaminothiazol-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)benzoxazole (BF-227),⁷³ and (¹¹C)-2-(6-[methylamino]pyridin-3-yl)-1,3-benzothiazol-6-ol (AZD2184).⁷⁴ It is clear that this type of PrP diagnostics related diseases has a great potential to be further developed and new imaging assays are still being looked for. Some of them could be based on quantum dots (QDs).

QDs as Alternative for Prion Detection

In the 1970s and early 1980s, understanding the photophysical properties of semiconductor structures was important for a broad range of computer and electronic applications. It was hypothesized that the physical properties of structures in an intermediate size range (between single atoms and bulk) could be tuned by alteration of size and shape. For electronics and computer applications, such a system allows to synthesize a large set of nanometer sized building blocks for constructing faster and smaller computer chips or more efficient light-emitting devices. It was soon realized that these nanometer sized structures called quantum dots (QDs) could have significantly much more applications. Nowadays a wide range of QDs utilizations are found in the area of biology, biochemistry and biomedicine. Besides the applications as simple sensors,^75-77 the main function of QDs based on their exceptional fluorescent properties in the biochemical and biomedical research area is their usage as unique fluorescent labels.^78-80 The utilization of QDs for various purposes is shown in Figure 2.

Figure 2. Applications of QDs in chemical, biomedical and diagnostic area. QDs (the large green crystal) can be applied for biosensing, biomarker mapping, real time cell growth monitoring, drug delivering, gene delivering, detection and diagnostics, molecular imaging, and targeted therapy.

The most important characterization of QDs dealing with their optical properties is usually provided by UV-VIS and photoluminescence spectroscopy which offer fast, non-destructive and contactless option. Besides monitoring the excitation, absorption and emission spectra which are usually applied for the calculation of quantum yield, band gap studies can be also performed by optical diffuse reflectance spectra measurement. The size of QDs can be calculated from absorption edges using Henglein empirical curve, which relates the wavelength of the absorption threshold to the diameter of QDs.⁸¹ In particular cases, FTIR spectroscopic measurements are also necessary.⁸² Raman spectroscopy, as one of the best non-destructive techniques, can be employed for QDs characterization as well, since it allows to probe the active optical phonon modes and to explore the confined electronic structure of QDs.^83,84

As mentioned before, the optical properties (fluorescence emission) of QDs can be fine-tuned by the QDs size which is a key parameter that determines the spectral position and purity of photoluminescence. QDs size and morphology (shape and structure) are generally calculated using conventional techniques like scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) studies. For these measurements, QDs are usually transformed to powder form either by simple drying of QDs solution or by precipitation (e.g. with ethanol), centrifugation and final drying. Besides these techniques, field flow fractionation, which belongs to high resolution liquid chromatography-like elution methods for separating and sizing, can be also successfully employed for QDs size distribution analysis.⁸⁵ The structure of QDs is usually analyzed by X-ray diffraction (XRD)⁸⁶ and the elemental composition of QDs can be studied by energy dispersive X-ray analysis (known as EDS, EDX, or EDAX).⁸² Other techniques, like inductively coupled plasma atomic emission spectrometry (ICP-AES), were used for analysis of QDs as well, namely for the metal ions content in QDs.⁸⁷ Recently, electrochemical methods were also applied to study the QDs behavior.^88-90

Biocompatibility

To use QDs in biology, it is extremely important to deal with the biocompatibility and toxicity. However, minimum studies have examined the toxicity of these nanomaterials.^91-93 Moreover, their toxicity evaluation is highly complicated, due to the diversity of materials. In sharp contrast to conventional hazardous materials, the attention has to be paid to the nanoparticle-specific problems, including the fact that the surface of nanomaterials is very active due to the large surface area and surface-to-volume ratio. In addition, it is necessary to exclude the effect of solubility and possible contamination which could also decrease the validity of any toxicity testing.⁹² In 2006, Hardman reviewed the toxicity of QDs as a function of physicochemical and environmental factors.⁹⁴ QDs size, charge, concentration, outer coating bioactivity (capping material, functional groups) as well as oxidative, photolytic, and mechanical stability have each been shown as determining factors of QDs toxicity.

To improve the biocompatibility, passivation or capping of QDs with a layer of ZnS or CdS is needed. ZnS or CdS improve the fluorescence quantum yield of the QDs and protect them against photo-oxidation, which is important for minimizing cytotoxicity and enhancing photostability. ZnS shell has larger band gap energy than CdSe, eliminating the core’s surface defect states. ZnS shell has also similar bond length to CdSe, minimizing the crystal-lattice strain and allowing the epitaxial growth. Even with advances in synthesis, obtaining biomedically useful QDs is still problematic due to differences in optical properties from batch to batch. From one synthesis to the next, QDs with different quantum yields and fluorescence spectra may be produced. Moreover, further functionalization is needed for incorporation of required chemical species.

Various surface modification techniques were developed to ensure the specific bio-conjugation (Fig. 3). This is usually achieved by modifying QDs with proteins, peptides, nucleic acids, or other biomolecules that mediate specific interactions with living systems. Surface engineering is thus crucial not only for tuning the fundamental properties of nanomaterials and for rendering them stable and soluble in different environments, but also for creating nanoparticle–biomolecule hybrids capable of participating in biological processes. Such hybrids should combine useful properties of both materials involved, i.e. the optical properties of nanocrystals and biological functions of ligands attached.⁷⁸ One of these strategies utilizes the biotin-avidin (respectively streptavidin and neutravidin) interaction known for its very high specificity. Modification of QDs by the streptavidin proved a very successful method evaluated in numerous publications.^78,95-98 Due to this success, streptavidin-QDs are nowadays also commercially available. In addition, biotin-functionalized QDs were developed to exploit the same interaction (Fig. 2).

Figure 3. (A) Differential pulse voltammograms of prion protein (PrP, 500 µg/mL) with additions of QDs in various volumes (460 µg/mL, quantified according to concentration of cadmium[II]). Electrochemical measurements were performed with AUTOLAB Analyzer (EcoChemie) connected to VA-Stand 663 (Metrohm), using a standard cell with three electrodes. The working electrode was a hanging mercury drop electrode (HMDE) with a drop area of 0.4 mm². The reference electrode was an Ag/AgCl/3M KCl electrode and the auxiliary electrode was a graphite electrode. Acetate buffer (0.2 M, pH 5) was used as the supporting electrolyte. For smoothing and baseline correction the software GPES 4.9 supplied by EcoChemie was employed. The amount of QDs was measured using DPV. Differential pulse voltammetric measurements were performed under the following parameters: start potential -1.5 V; end potential 0 V; modulation time 0.057 s, time interval 0.2 s, step potential of 1.05 mV/s, modulation amplitude of 250 mV, Eads = 0 V. All experiments were performed at room temperature (20 °C). The DPV samples analyzed were deoxygenated prior to measurements by purging with argon (99.999%) saturated with water for 120 s. (B) In vivo images of 10 µl (460 µg/mL) of CdTe QDs with prion protein (PrP, 500 µg/mL) applied 1 cm under skin of chicken thigh. The image of chicken leg without any injection was subtracted. The fluorescence imaging was performed by In vivo Xtreme system by Carestream. This instrument is equipped with 400 W xenon light source and 28 excitation filters (410–760 nm). The emitted light is captured by 4MP CCD camera. In this experiment, 410 nm was used as an excitation wavelength and the emission was measured at 535 nm. The exposition time was 5 s. The other parameters were set as follows: Bin, 2 × 2; field of view, 12 cm; fStop, 1.1. (C) Scheme of complex of QDs and antibody with prion protein and the possible using of such prion protein sensing in brain tissue.

In vitro applications

The conditions of in vitro experiments are artificial and simulate what might happen in vivo. In the last decade, surface engineering and biofunctionalization techniques have transformed semiconductor nanocrystals into complex cellular probes capable of interaction with biomolecules and direct participation in biological processes. In 1998, two seminal scientific papers demonstrated for the first time that semiconductor nanoparticles could be made water-soluble and used as biological imaging probes.^99,100 One approach utilized encapsulation of QDs in silica shell in order to produce QDs for a single-excitation dual-color cell staining.⁹⁹ When derived with trimethoxysilylpropyl urea and acetate groups, green QDs preferentially labeled the cell nucleus, and when modified with biotin, red QDs labeled F-actin filaments pretreated with phalloidin-biotin and streptavidin. The second paper was the first to demonstrate the ligand-exchange approach to QDs water solubilization.¹⁰⁰ Subsequent conjugation of transferrin produced QDs probes that were endocytosed by live HeLa cells, resulting in the punctate cell staining, while IgG bioconjugates were used in an aggregation-based immunoassay. Since then, many surface engineering techniques for QDs solubilization and biofunctionalization have been developed, enabling the application of QDs probes with specific design. Such probes have found their use in a variety of in vitro applications, such as histological evaluation of cells and tissue specimens, single molecule detection and real-time tracking, long-term live-cell imaging, and study of intracellular processes. Moreover, QDs can be employed as optical labels that probe dynamic biological processes, such as biocatalysed reactions or structurally induced biomolecular transformations using fluorescence resonance energy transfer (FRET) or electron-transfer quenching as photophysical probing mechanisms (Fig. 2).

Recent achievements in merging nanoparticle encapsulation and bioconjugation steps and design of pre-functionalized surface coatings promise to provide more compact, stable, and biocompatible nanoparticles with controlled density and orientation of ligands attached. Amphiphilic polymers with a maleic anhydride backbone are being actively explored for this purpose. In organic anhydrous solvents, such polymers encapsulate trioctylphosphine-coated QDs and introduce reactive anhydride groups on the surface. In basic aqueous buffers, anhydride rings are quickly hydrolyzed, yielding negatively charged carboxylic acid groups and rendering QDs water soluble.¹⁰¹ More importantly, anhydride groups are highly reactive toward amine-containing molecules, thus allowing covalent conjugation of various biomolecules to the polymer chains without the need for post-encapsulation modification.^102,103

The choice of bioconjugation approach depends on the availability of ligands with suitable functional groups and on specific application requirements. However, common design criteria involve preserved QDs photo-physical properties and ligand biofunctionality, controlled ligand orientation and binding stoichiometry, compact probe size, and good stability in physiological environment. As these criteria can be satisfied in only few specific cases, improvement of existing bioconjugation techniques and design of novel application specific water solubilization and bioconjugation approaches remain an active area of research. With the development of stable and biofunctional QDs probes, these materials will become the nanoscience building blocks with flexible properties that could be further optimized for specific applications, including biomedical imaging, detection, and nano-therapeutics.¹⁰⁴

Electrochemical detection as a sensitive technique in combination with QDs has been already used for numerous applications. However, QDs have not been sufficiently analyzed by voltammetry with respect to employment these easy to use and repeatable methods for QDs characterization and quantification. Therefore, CdTe QDs were characterized using cyclic and differential pulse voltammetry. The obtained peaks were identified and detection limit (3 S/N) was estimated down to 100 fg/mL. Based on convincing results, new way how to study the stability and quantify QDs was suggested. The approach was further utilized for testing of QDs stability.⁵ Interaction of PrP with CdTe QDs by voltammetry seems to be a new and extremely sensitive tool for sensing of these proteins. Primarily, fluorescent and electrochemical properties of QDs were characterized. Further, electrochemical study of their interactions was performed to find the most suitable conditions for sensitive detection of PrP (Fig. 3A). Detection limit (3 S/N) was estimated as 1 fg in 5 µL.¹⁰⁵ This makes labeling of proteins with QDs of great importance due to easy applicability and possibility to use in miniaturized devices. Based on our results, it can be concluded that QDs-PrP complex is stable and can be quantified in extremely low amounts. The stability of complex in vivo was also proven (Fig. 3B). This should open new possibilities how to determine the presence of these proteins in a tissue (Fig. 3C), on surgical equipment and on other types of materials which could be contagious (Fig. 4A–D).

Figure 4. Sensing of prion protein complex with quantum dots on the surface of a scalpel. (A) photo of a scalpel, (B) fluorescent image of scalpel, (C) fluorescent image of scalpel with prion protein-QDs complex, and (D) detail of fluorescent image of scalpel with prion protein-QDs complex. QD were prepared according to Duan et al.¹⁰⁶ Cadmium chloride solution (CdCl₂, 0.04 M, 4 ml) was diluted to 42 ml with ultrapure water, and then trisodium citrate dihydrate (100 mg), Na₂TeO₃ (0.01 M, 4 ml), MPA (119 mg), and NaBH₄ (50 mg) were added successively under magnetic stirring. The molar ratio of Cd²⁺/MPA/Te was 1:7:0.25. Ten ml of the resulting CdTe precursor was put into a Teflon vessel. CdTe QDs were prepared at 95 °C for various times according to desired color (10 min, green; 30 min, yellow; 120 min, red) under microwave irradiation (400 W, Multiwave3000, Anton-Paar GmbH). After microwave irradiation, the mixture was cooled to 50 °C and the CdTe QDs sample was obtained. Purification of CdTe QDs was performed using isopropanol condensing. The CdTe QDs was mixed with isopropanol in ratio 1:2 and then centrifuged for 10 min at 25 000 rpm (Eppendorf centrifuge 5417R). Precipitate (pure CdTe QDs) was than resuspended to initial volume of Tris Buffer pH 8.5. The fluorescence imaging was performed by In vivo Xtreme system by Carestream. This instrument is equipped with 400 W xenon light source and 28 excitation filters (410–760 nm). The emitted light is captured by 4MP CCD camera. In this experiment, 410 nm was used as an excitation wavelength and the emission was measured at 535 nm. The exposition time was 5 s. The other parameters were set as follows: Bin, 2 × 2; field of view, 12 cm; fStop, 1.1.

In vivo applications

Even though some attempts for optical imaging of prion based diseases have been made, these were mostly in in vitro mode.^107,108 A new and exciting direction of research using QDs is their application as contrast agents for in vivo imaging.^109-112 The characterization and analysis of biomolecules and biological systems in the context of intact organisms is known as in vivo research. The in vivo approach involves experiments performed in the large system of an experimental animal body. Organic fluorophores and chemiluminescence probes are currently the most commonly used optical probes for animal imaging. However, the lack of available probes that emit in NIR emission range (>650 nm) cause a significant limitation of these optical contrast agents. The NIR emitting window is appealing for biological optical imaging because of low tissue absorption and scattering effects in this emission range. The range of NIR optical window for animal imaging is typically set at 650–900 nm. Since the optical properties of QDs can be tuned by size and composition, it is possible to prepare series of NIR-emitting QDs for animal imaging.¹¹³ CdTe, CdTeSe, InPAs, PbS, and PbSe have been successfully synthesized with NIR emission.^114,115 For most in vivo imaging investigations , QDs are usually directly injected into the living animal intravenously or subcutaneously and thereby are delivered into the bloodstream.

Future Prospects

QDs, tiny light-emitting nanocrystals, have emerged as a new promising class of fluorescent probes for biomolecular and cellular imaging. In comparison with organic dyes and fluorescent proteins, QDs possess unique optical and electronic properties such as size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. The biomedical applications of nanoparticles are rooted in the advanced functional design and have been realized in preclinical experimental diagnosis. As compared with the imaging in vitro, QDs imaging in vivo faces different challenges, caused by the increase of complexity of multicellular organism. There are four main kinds of in vivo imaging applications with QDs: (1) biodistribution of QDs, (2) vascular imaging, (3) tracking of QDs, and (4) tumor imaging (Fig. 2). Utilization of nanomaterials, particularly nanoparticles for the in vivo monitoring of brain diseases is one of near future appealing applications.¹¹⁶ The incorporation of contrast agents into the cells can be done by either phagocytosis or conjugation of the contrast agent to the cell surface via antibody interaction with the receptor. In vivo imaging of amyloid plaques may be useful for the evaluation and diagnosis of AD patients (Fig. 3).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Financial support from CEITEC CZ.1.05/1.1.00/02.0068, DOC CEITEC.01/2012, Grant Agency of the Czech Republic under the contract GACR 102/13-20303P and IGA TP1/2013 (EA 3b) is highly acknowledged. The author Sobrova P is “Holder of Brno PhD Talent Financial Aid”.

10.4161/pri.26524