|Year : 2016 | Volume
| Issue : 1 | Page : 21-28
Quantum Dot-based Immunohistochemistry for Pathological Applications
Li Zhou1, Jingzhe Yan2, Lingxia Tong3, Xuezhe Han4, Xuefeng Wu5, Peng Guo6
1 Department of Gynecologic Oncosurgery, Jilin Provincial Cancer Hospital, Changchun, Jilin, China
2 Department of Colorectal Surgery, Jilin Provincial Cancer Hospital, Changchun, Jilin, China
3 Department of Ultrasonography, Jilin Provincial Cancer Hospital, Changchun, Jilin, China
4 Department of Neurosurgery, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China
5 Clinical Laboratory, Jilin Provincial Cancer Hospital Changchun, Changchun, Jilin, China
6 Department of Biomedical Engineering, Shenzhen University, Shenzhen, Guangdong, China
|Date of Submission||29-Jul-2015|
|Date of Acceptance||05-Jan-2016|
|Date of Web Publication||26-Feb-2016|
Department of Biomedical Engineering, Shenzhen University, 3688 Nanhai Road, Shenzhen 518060, Guangdong
Source of Support: None, Conflict of Interest: None
Quantum dots (QDs) are novel light emitting semiconductor nanocrystals with diameter ranging from 2 to 20 nm. In comparison with traditional organic dyes and fluorescent proteins, QDs possess unique optical properties including extremely high fluorescence efficiency and minimal photobleaching which make them emerge as a new class of fluorescent labels for molecular imaging and biomedical analysis. Herein, recent advances in fundamental mechanisms and pathological applications of QD were reviewed.
Keywords: Cancer biomarker, immunohistochemistry, pathology, quantum dot
|How to cite this article:|
Zhou L, Yan J, Tong L, Han X, Wu X, Guo P. Quantum Dot-based Immunohistochemistry for Pathological Applications. Cancer Transl Med 2016;2:21-8
|How to cite this URL:|
Zhou L, Yan J, Tong L, Han X, Wu X, Guo P. Quantum Dot-based Immunohistochemistry for Pathological Applications. Cancer Transl Med [serial online] 2016 [cited 2019 Jan 17];2:21-8. Available from: http://www.cancertm.com/text.asp?2016/2/1/21/177562
| Introduction|| |
Quantum dots (QDs), also called semiconductor nanocrystals, are new types of light-emitting fluorophores with inorganic core-shell structure. The size of QDs ranges from 2 to 20 nm, and QDs are mainly composed of atoms from Group II to VI elements in the periodic table. The most commonly used inner core materials for QDs are CdSe and CdS and outer shell materials are ZnS, CdS, and ZnSe.  The concept of QD originates from the discovery of quantum confinement effect in the early 1980s. ,, In 1981, Ekimov and Onoshchenko  published their paper entitled, "Quantum size effect in three-dimensional microscopic semiconductor crystals" in JETP Letters, which represents the discovery of CuCl QDs in transport dielectric matrix. Later in 1985, Rossetti et al. reported the same phenomenon in colloidal solution. The official name of "quantum dot" was introduced by Reed et al. in 1988, with a definition of semiconducting crystalline fluorophore whose radius is below its exciton Bohr radius.
When a QD is excited by photons with energy higher than its semiconductor band gap energy, the QD will absorb the energy and excite one electron from its valence band into its empty conduction band, leaving a hole of opposite charge in the valence band. This electron and its hole attract each other by Coulomb forces and form an exciton. Because the radius of QD is less than its Bohr radius, the exciton from QD is spatially squeezed and confined within the QD radius, which leads to a quantization of the energy levels of valence and conduction bands within the QD according to Pauli's exclusion principle.  This procedure is called quantum confinement effect which provides QD with unique optical and electronic properties.
With rapid developments in bionanotechnology, QDs have emerged as a promising multifunctional tool in biomedical applications, including molecular imaging, , drug delivery, , and immunohistochemical (IHC) staining.  For example, Chen et al.  developed a QD-based magneto-fluorescent supernanoparticle to achieve in vivo multiphoton imaging for brain tumor. QDs have also been used as an efficient drug/siRNA delivery vehicle. Considering the cytotoxic effects of QD's, QD-based IHC (QD-IHC) is performed on the tissue samples at in vitro or ex vivo levels, which may represent the potential clinical applications of QDs.
In this review, we provide an overview of recent advances in the development and application of QDs in IHC for clinical diagnosis. We discuss several topics in this review, including optical properties, cytotoxicity, bioconjugation, and pathological applications. We also discuss current limitation and future perspective for QD-IHC.
| Optical Properties of Quantum Dots|| |
In comparison with traditional organic dyes and fluorescent proteins, QDs demonstrate following unique optical advantages. (1) As a semiconducting nanomaterial, QD can be readily excited by light within a wide range of wavelength from ultraviolet (UV) to infrared (IR) and emits fluorescence when the excited electron moves back to valence band from conduction band. Due to the quantum confinement effect, QD emits a stronger fluorescence within a tightly defined spectrum with an emission width of 15-30 nm,  which allows different QDs to facilitate multiplex immunostaining in the pathological applications.  (2) The wavelength of emitted fluorescence from QD is closely associated with the radius of QDs. Thus, the fluorescent color of QDs can be readily tuned by controlling its diameter during QD synthesis.  Generally, the wavelength of fluorescence emitted decreases with the size of the QD. Bruchez et al.  demonstrated that QDs can achieve emission wavelength of 400 nm to 2 mm in the peak emission, with typical emission widths of 20-30 nm. (3) QDs can exhibit long fluorescence lifetime and low photobleaching properties. In the core-shell structure of QDs, the core materials are mainly responsible for the fluorescence quantum yield, and the shell materials are used to protect the core from oxidation, increase its photostability, and improve its quantum yield. Polymers such as polyethylene glycol (PEG) are often coated on QD to improve its biocompatibility in biomedical applications. 
| Cytotoxicity of Quantum Dots|| |
In spite of their unique optical properties, an undeniable health concern of QDs is their cytotoxicity due to elemental composition. Most QD core materials consist of a variety of toxic metal complexes including CdSe, InAs, and ZnS which are known to be harmful to vertebrates. , Many in vitro studies have suggested that QDs, even with polymer coatings, are still cytotoxic to cell cultures.  Lovrić reported that mercaptoacetic acid (MPA)/cysteine coated CdTe QDs were cytotoxic to rat pheochromocytoma cells (PC12) by inducing the generation of cytotoxic reactive oxygen species.  Hoshino et al.  also reported that CdSe/ZnS QD-induced DNA damage in WTK1 cells at a concentration of 2 μmol/L. Polymer coatings, such as PEG, mercaptoundecanoic acid, and sheep serum albumin, have been investigated to improve QD's biocompatibility temporally, ,, but this method cannot fundamentally resolve the cytotoxic effects of QDs since the QD components will eventually leak from the polymeric coatings and are exposed to in vivo environment. Many other factors may also contribute to the cytotoxicity of QDs, such as particle size, surface charge, and dosage. , The applications of QD for in vivo bioimaging are significantly limited due to its potential acute and chronic cytotoxicity. In the context of pathological applications, most analyses of cell and tissue biomarkers are performed at in vitro or ex vivo levels. Thus, the cytotoxicity of QDs is no longer a concern for the pathological analysis which makes QD a promising candidate for IHC staining agents.
| Bioconjugation of Quantum Dots|| |
Regular QDs have nonspecific binding to proteins, cellular membranes, or extracellular matrices. Therefore, naked QDs are indiscriminately taken up by various types of cells.  PEG is commonly coated on QDs to minimize its nonspecific binding with cells.  To use QDs as fluorescent probes for specific cellular or tissue biomarkers in IHC staining, biomarker-specific molecules have to be conjugated on QDs through surface functionalization. Common biomarker-specific molecules are mainly antibodies, natural ligand proteins, and engineered peptides or nucleotides. ,, In general, there are three types of bioconjugation strategies for QDs: (1) Carbodiimide, (2) thioether, and (3) biotin-avidin conjugations.
This is the most common strategy to covalently conjugate QDs with target molecules such as antibodies. Firstly, it requires functionalizing QDs with carboxylic acid. Then, the carboxylic acid group on QD surface will be activated in situ by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The activated ester reacts with primary amines presenting on antibodies or peptides to form a stable amide bond. This conjugation strategy is straightforward and efficient. This conjugation reaction can occur at ambient temperature and atmosphere, neutral pH conditions, and within a short period of reaction time. A representative study is that Chan and Nie successfully used carbodiimide conjugation to functionalize CdSe/ZnS QDs with transferrin [Figure 1]a and b].  One major problem of carbodiimide conjugation is that antibodies are randomly attached to QD surface due to multiple amines presenting on the antibodies or peptides, which may sterically hinder the binding affinity of antibody conjugated QDs toward biomarker proteins in tissues. The linkage spacer distance is zero between antibodies and QDs, which may further increase the steric hindrance.
|Figure 1. Quantum dots developed for cellular imaging. (a) Schematic of a ZnS-capped CdSe QD that is covalently coupled to a protein by mercaptoacetic acid. (b) Transmission electron microscopy of quantum dot transferrin conjugates. Scale bar, 100 nm. (c) Luminescence images of cultured HeLa cells that were incubated with quantum dot-transferrin conjugates. Cell diameter, ~10 μm. Reproduced with permission.28 Copyright 1998, The American Association for the Advancement of Science. (d) Fluorescent image of mouse 3T3 fibroblast cells labeled with red and green fluorescence CdSe-CdS quantum dots. Reproduced with permission.31 Copyright 1998, The American Association for the Advancement of Science|
Click here to view
To gain the control over the orientation of antibodies conjugated on QD surface, an effective strategy was developed via producing controllable thiolated antibodies by reducing disulfide bonds in antibodies. Then, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) is used to catalyze amine functionalized QD to react with thiolated antibodies. Primary amine group on QD is first activated by SMCC to form a reactive maleimide group, and then this newly-formed maleimide group covalently reacts with thiol group on antibodies to form a stable thioether bond and bridge each other. Another advantage of thioether conjugation is that linkage spacer can be added at different lengths or even cleavable groups into linkers. For example, Pathak et al.  demonstrated that using SMCC linker to conjugate functional antibodies to QDs was viable.
Another common QD conjugation strategy is conjugating biotinylated antibodies with avidin-coated QDs.  This strategy uses a specific interaction between biotin and avidin and can generate a potent and stable antibody-QD linkage. Similar to carbodiimide conjugation, the drawback of biotin-avidin conjugation is that it has no control over the orientation of antibodies conjugated on QD surface. This is because there are multiple biotinylation sites presented on each antibody and can indiscriminately interact with avidin-coated QDs.
| Pathological Applications of Quantum Dots|| |
Cellular imaging by quantum dots
QDs were first introduced into cellular labeling by Chan and Nie  and Bruchez et al.  independently in 1998. Bruchez et al. reported that they successfully used SiO 2 coated CdSe/CdS QDs to label mouse fibroblast 3T3 cells for fluorescent labeling [Figure 1]d]. Chan and Nie  reported that they covalently conjugated ZnS-capped CdSe QDs with transferrin protein using MPA, and utilized the specificity of transferrin conjugated on QDs to selectively bind with HeLa cells [Figure 1]a-c].  In 2003, Wu et al.  used QDs linked to immunoglobulin G and streptavidin to label the breast cancer marker, human epidermal growth factor receptor 2 (HER2), on the surface of fixed and live cancer cells, to stain actin and microtubule fibers in cytoplasm, and to detect nuclear antigens inside the nucleus. They found that all labeling signals are specific for the intended targets and are brighter and considerably more photostable than comparable organic dyes. These pioneer studies indicate that targeted QDs can effectively bind with biomarkers in cellular imaging and offer substantial advantages over traditional fluorescent dyes.
Single biomarker quantum dot-immunohistochemical staining
In addition to cellular imaging, the application of QDs in tissue sample staining, especially IHC may have more clinical values in disease diagnosis. IHC is the integration of anatomical, immunological, and biochemical analyses to discrete diseased components in tissue samples, which is achieved by tagging target antigens with specific antibodies labeled with dyes or fluorophore. IHC is widely used in the pathology department of hospitals as a tool to evaluate and diagnose following histopathological assessments. , The main advantage of IHC is its capability to retain the morphology of the tissue, which can be used to predict its response to specific treatments. The major limitation of traditional IHC is that the staining severely suffers from photobleaching under constant illumination because of the usage of organic fluorescent dyes (such as fluorescein, cyanide, and Alexa Fluor dyes). The exceptional optical properties of QDs over traditional methods can significantly improve the sensitivity and stability of IHC results, which can be a breakthrough in developing more efficient IHC tools. IHC is also an ex vivo process which can avoid the potential cytotoxicity of QDs. Because multiple different QDs can be simultaneously excited by the same UV-light, QDs have a dramatic advantage in multiplex IHC with more profound impacts on clinical diagnosis.
The first application of QD in IHC was reported by Ness et al.  in 2003. They stained MAP2 in mouse embryo sections with CdSe QDs via streptavidin-biotin interaction, and results indicated that QD-IHC is significantly more sensitive than traditional IHC staining with organic fluorescent dye Cy3.  In 2004, Wang et al.  successfully used QDs to detect the ovarian carcinoma marker CA125 in both tissue section and xenograft sections. They also compared the specificity, fluorescent intensity, and photostability of QD with conventional organic dye and FITC, and their results confirmed that QDs possess substantial advantages over organic dyes. Later this year, Colton et al.  reported that PMP70 antibody conjugated QDs can be used to stain and quantify the peroxisomes proliferation in rat and monkey livers with fibrates. After these successful proof-of-principle demonstrations, QD-IHC draws enormous attention from oncologists and pathologists due to its exceptional optical properties. A number of single color QD-IHC studies have been conducted to detect cancerous cells in a variety of tumor tissues via common cancer biomarkers. Wu et al.  utilized QDs to localize the breast cancer cell surface marker HER2, cytoskeleton fibers, and nuclear antigens in fixed cells, live cells, and tissue sections. Wang et al.  developed QD-labeled antibodies for rapid visualization of epidermal growth factor receptor (EGFR) expression in human brain tumor cells and surgical frozen section slides of glioma tissue. They demonstrated that QD-IHC was a quick and accurate method for characterizing cancer biomarker in human brain tumor tissues. Cheng et al. compared QD-IHC with fluorescent dye-IHC in detecting the expression of prostate stem cell antigen (PSCA) in bladder tumor tissues. The results revealed that these two methods had similar sensitivity in the differential display of PSCA expression correlated with tumor stage and grade, but optical properties of QDs demonstrated obvious advantages over organic molecular dyes in staining high autofluorescent tissue region, such as retina (lipofuscin and melanin). Petty et al.  investigated QD-IHC to stain human retina tissue via targeting RPE65 expression. Qu et al.  described QD-IHC detection of EGFR gene mutations in nonsmall cell lung cancers using mutation-specific antibodies. Based on these single biomarker QD-IHC studies, we could readily conclude that QD-IHC is generally brighter, significantly more sensitive and photostable than current conventional IHC, which suggests QDs may represent the next generation of fluorescent probe for clinically used IHC.
Multiplexed quantum dot-immunohistochemical staining
In comparison with organic fluorescent dyes, QDs can be excited by light within a wide range of wavelength from UV to IR and emit stronger fluorescence within tightly defined peaks. This unique property allows QDs with different emission spectrums to facilitate multiplex IHC staining in pathological applications. Application of this multiplex method enables investigators to explore the clinically relevant multidimensional cellular interactions that underlie diseases, simultaneously. In 2006, Fountaine et al.  used QDs to determine the expression and spatial distribution of a panel of clinically relevant cellular targets (CD20, MIB-1, MUM-1, BCL-6, etc.,) in tonsil and lymphoid tissues. Wu et al.  reported the QD bioconjugates targeting mutated p53 and early growth response protein to examine prostate cancer tissues, which indicates a multiplex and quantitative strategy of clinical biomarker study.
With the development of multiplexed QD-IHC techniques, Yezhelyev et al.  reported the in situ molecular profiling of five breast cancer biomarkers in both cultured human breast cancer cells and single paraffin-embedded clinical tissue sections. They found QD-IHC staining intensities of estrogen receptors (ERs), progesterone receptors, and HER2 receptors correlated closely with the results from organic dye-based IHC, Western blotting, and fluorescent in situ hybridization. Chaudry et al.  harnessed QD-IHC to develop and validate a panel of clinical biomarkers of colorectal cancer and assess the cancer risk based on quantitative QD and molecular profiling. Sweeney et al.  reported the simultaneous multiplex QD-IHC staining with three antibodies. CD34, cytokeratin 18, and cleaved caspase 3 were triplexed in tonsillar tissue. Caldwell et al.  used multiplexed QD-IHC to target two biomarkers (MDM-2 and B-actin) in the renal cell carcinoma tissue samples that are distinguishable from adjacent normal tissue samples. Chen et al.  compared QD-IHC and conventional IHC for the detection of caveolin-1 and proliferating cell nuclear antigen in the lung cancer tissue microarray [Figure 2] and demonstrated that QDs exhibited a much better photostability, broader excitation spectrum, and longer fluorescence lifetime. Nie laboratory has developed QD-IHC staining techniques by mapping a panel of 4 protein biomarkers (E-cadherin, high-molecular-weight cytokeratin, p63, and α-methylacyl CoA racemase) in human prostate cancer tissue specimens and demonstrated that QD-IHC staining can successfully characterize structurally distinct prostate glands and single cancer cells within the complex microenvironments of radical prostatectomy and needle biopsy tissue specimens.  They also further applied multiplexed QD-IHC staining of CD15, CD30, CD45, and Pax5 to characterize low abundant cancer cells in human Hodgkin's lymphoma tissues [Figure 3].  Notably, they performed QD-IHC in tissues from 6 confirmed Hodgkin's lymphoma patients, 2 suspicious lymphoma cases, and 2 patients with reactive lymph nodes (but not lymphoma), and their results demonstrated a distinct QD staining pattern (CD15 positive, CD30 positive, CD45 negative, and Pax5 positive) to differentiate Hodgkin's lymphoma from benign lymphoid hyperplasia.
|Figure 2. Digital images of single biomarker quantum dot - immunohistochemical and conventional immunohistochemical staining of caveolin-1 in the lung cancer tissue. (a and c) quantum dot immunohistochemical, (b and d) conventional immunohistochemical (a and b, ×100; c and d, ×200). Reproduced with permission.47 Copyright 2009, Springer|
Click here to view
|Figure 3. Quantum dots developed for multiplexed immunohistochemical staining. (a) Schematic illustration of multiplexed quantum dot tissue staining with two primary antibodies from two animal species that recognize two tissue antigens. (b and c) Multiplexed quantum dot staining images of HRS malignant cells and infiltrating immune cells on lymph node tissue specimens of a Hodgkin's lymphoma patient. Scale bars: (b) 100 ìm; (c) 10 ìm. Reproduced with permission.49 Copyright 2010, American Chemical Society|
Click here to view
With the development of quantitative QD-IHC technologies, it is possible to quantify the molecular biomarker expression of patient's pathological tissues and correlate them with their clinical prognosis, which translates the QD-IHC research from scientific research into clinical diagnosis. For example, Chen et al.  used QDs to study the breast tumor heterogeneity and simultaneous imaging of ER and HER2 to understand their interaction during the process of evaluation of heterogeneous breast cancer. In another study, Matsuno et al.  showed a three-dimensional imaging of the intracellular localization of growth hormone and prolactin and their mRNA in rat pituitary glands, using QDs (for protein detection) and HRP-DAB (for RNA detection) via confocal laser scanning microscope. Matsuno's research provides important insights into the relationship of protein and mRNA synthesis/localization. Orndorff et al.  successfully extended QD-IHC staining to visualize and quantify nicotinic receptors expression in mouse brain neuromuscular synapses which allow the direct assessment of the presence and mobility of neurotransmitter receptors in native tissue. Li laboratory has published a number of papers about using QD-IHC to study breast cancer patient prognosis. ,,,, They developed and validated the QD-IHC protocols for quantifying HER2 expression in an accurate, sensitive, and convenient approach on breast cancer specimens.  They also used QD-IHC to characterize the breast tumor heterogeneity and successfully identified a new subtype of breast cancer with distinctly different 5-year disease-free survival rate in comparison with existing breast cancer subtypes.  Xue et al.  used QD-IHC to characterize caveolin-1 expression and location in tongue squamous cell carcinoma. Their findings indicated that increased caveolin-1 expression levels can promote the development of tongue squamous cell carcinoma. Chen et al.  used QD-IHC to characterize EZH2 and P53 protein in biopsy tissues of 168 patients with cervical squamous cell carcinoma. Their findings showed increased EZH2 and P53 expression in patients was closely associated with the lymph node metastasis of cervical cancer.
Although QD-IHC demonstrates tremendous success in pathology applications, limitations still exist and need further evaluation. Korzhevskii et al.  revealed a number of disadvantages of QD-IHC such as low stability on prolonged storage, poor reagent preservation, inability to retain fluorescence in stained specimens, and incompatibility with commercially available media for embedding specimens. These limitations prevent the widespread use of nanocrystals in IHC.  In another study, Yu et al.  also found unexpected and subtle changes to QD emission spectra when multiplexed (vs. when not multiplexed) which resulted in complicated unmixing of the QD signals.
One major bottleneck for QD-IHC is the nonspecific binding of QDs with cellular membranes. Thus, many coating materials have been investigated to minimize nonspecific QD binding. For example, Nie laboratory developed hydroxylated QDs that can facilitate a 140-fold reduction in nonspecific binding compared with that of traditional carboxylated QDs and a significant 10- to 20-fold reduction compared with that of PEG- and protein-coated QDs.  The fast-developing peptide engineering technique provides more specific and less immunogenic targeting ligands, which can further minimize the nonspecific staining of QD-IHC. For example, small bivalent engineered antibody fragments, cys-diabodies, have been engineered to conjugate with QDs as targeting ligands, and successfully applied in IHC with HER2 and PSCA expression.  Sukhanova et al.  engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) in a highly oriented manner. These sdAbs conjugated QDs demonstrated excellent specificity inflow cytometric quantitative discrimination of CEA-positive and CEA-negative tumor cells.
Many new QD-IHC techniques have been continuously developed to solve the existing problems. For example, Li-Shishido et al. reported β-mercaptoethanol and glutathione can effectively inhibit the blinking and bleaching of the QDs via inhibiting their "off-state."  Xiao et al. developed a novel QD-IHC that utilized chicken IgY antibody for high sensitivity and specificity relative quantitation of HER2 and telomerase biomarkers in solid tumor tissue arrays.  Zhou et al. reported a simple, one-pot synthesis of low-toxicity zinc sulfide immune-QDs which allow shorter synthesis time under mild condition (37°C). 
| Future Perspective|| |
To date, QD-IHC has achieved great development. However, the translation of QD-IHC from bench to bedside is still at the exploratory stage. In the next several years, QD-IHC may bring impacts in the following fields: (1) QD-IHC combined with other diagnostic techniques may provide precise and quantitative diagnosis. One representative example is the study by Xing et al.,  who prepared a targeting paramagnetic-fluorescent double-signal molecular nanoprobe for CRC in vivo magnetic resonance imaging diagnosis and subsequent biopsy. In another study, Ferrara et al. described QDs and two-photon excitation laser scanning microscopy to study three-dimensional morphology of human coronary arteries and mouse aortas, which is crucial to define the interactions of endothelial inflammatory markers and quantify the effects of different interventions on the endothelium. Due to the semiconductor nanocrystal nature of QDs, Killingsworth et al.  demonstrated that QD can be visualized by both light and electron microscopy. (2) QD-IHC is also a powerful tool to study pathological processes and functions of biological systems. Rieger et al. investigated QDs and GFP to co-label blood vessels of the vasculature and major axon tracts of the nervous system in the liver and fixed transparent zebrafish models. QDs had also been demonstrated to stain immune cells. Zahavy et al. developed a QD-based, double IHC technique for the simultaneous detection of T- and B-cells in paraffin-embedded mice spleen and axillary's lymph node tissues. Au et al.  used QDs to detect the breast cancer margins ex vivo within 30 min of tumor excision, indicating its potential as an accurate intraoperative margin assessment method. It is worth noting that using QD to quantify biomarker expression is not limited to tissue specimens and can be also applied to biological fluids. Hu et al.  developed QD-based microfluidic protein chip for ultrasensitive and multiplexed detection of cancer biomarkers directly in serum. (3) QD-IHC can be extended to visualize and quantify specific gene expression in tissues. Tholouli et al.  developed a series of QD-based oligonucleotide probes for myeloperoxidase, bcl-2, survivin, and XIAP and investigated them in spectral imaging analysis with formalin-fixed paraffin-embedded human biopsies of acute leukemia and follicular lymphoma, which allows quantitative characterization of multiple gene expression using nonbleaching fluorochromes. Naidoo et al.  used QD-IHC to detect YY1 protein expression levels in human follicular lymphoma (non-Hodgkin's lymphoma) and identified YY1 protein as a strong predictor of follicular lymphoma prognosis. Chen et al.  successfully used QD-IHC to detect Epstein-Barr virus in human nasopharyngeal carcinoma specimens via targeting EBV-encoded small RNA. In addition to biomarker protein/gene detection in tissue samples, QD-IHC staining has also been explored to detect pathogenic bacteria. Zhao et al.  combined QD-IHC staining to simultaneously detect three food-borne pathogenic bacteria, Salmonella typhimurium, Shigella flexneri, and Escherichia coli, which expand the application of QD-IHC from clinical diagnosis to food contamination detection.
| Conclusion|| |
QD-IHC is currently one of the most important and clinically relevant applications in QD research. QD-IHC offers a broad range of advantages to improve the prognostic evaluations. On one hand, QD-IHC exhibits exceptional optical properties over traditions fluorescent dyes via high and stable fluorescence with minimal photobleaching, giving them prime advantage in IHC staining. On the other hand, owing to the fast development of bioconjugation techniques, QD can be readily functionalized with various targeting ligands, making multiplex staining possible. Combining QD's advantages, we believe that in the near future, QD-IHC techniques will revolutionize the field of pathology by significantly increasing detection accuracy, sensitivity, and efficiency and can eventually improve the diagnosis-related clinical outcome.
Financial support and sponsorship
The study was supported by National Natural Science Foundation of China (81501572).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Dabbousi BO, Rodriguez-Viejo J, Mikulec FV, Heine JR, Mattoussi H, Ober R, Jensen KF, Bawendi MG. (CdSe) ZnS core - Shell quantum dots: synthesis and characterization of a size series of highly luminescent nanocrystallites. J Phys Chem B
1997; 101 (46): 9463-75.
Ekimov AI, Onoshchenko AA. Quantum size effect in three-dimensional microscopic semiconductor crystals. JETP Lett
1981; 34 (6): 345-9.
Rossetti R, Hull R, Gibson JM, Brus LE. Excited electronic states and optical spectra of ZnS and CdS crystallites in the ≈ 15 to 50 Å size range: evolution from molecular to bulk semiconducting properties. J Chem Phys
1985; 82 (1): 552-9.
Reed MA, Randall JN, Aggarwal RJ, Matyi RJ, Moore TM, Wetsel AE. Observation of discrete electronic states in a zero-dimensional semiconductor nanostructure. Phys Rev Lett
1988; 60 (6): 535-7.
Reimann SM, Manninen M. Electronic structure of quantum dots. Rev Mod Phys
2002; 74 (4): 1283-342.
Santra S, Dutta D, Walter GA, Moudgil BM. Fluorescent nanoparticle probes for cancer imaging. Technol Cancer Res Treat
2005; 4 (6): 593-602.
Chen O, Riedemann L, Etoc F, Herrmann H, Coppey M, Barch M, Farrar CT, Zhao J, Bruns OT, Wei H, Guo P, Cui J, Jensen R, Chen Y, Harris DK, Cordero JM, Wang Z, Jasanoff A, Fukumura D, Reimer R, Dahan M, Jain RK, Bawendi MG. Magneto-fluorescent core-shell supernanoparticles. Nat Commun
2014; 5: 5093.
Derfus AM, Chen AA, Min DH, Ruoslahti E, Bhatia SN. Targeted quantum dot conjugates for siRNA delivery. Bioconjug Chem
2007; 18 (5): 1391-6.
Sharei A, Cho N, Mao S, Jackson E, Poceviciute R, Adamo A, Zoldan J, Langer R, Jensen KF. Cell squeezing as a robust, microfluidic intracellular delivery platform. J Vis Exp
2013; (81): e50980.
Ness JM, Akhtar RS, Latham CB, Roth KA. Combined tyramide signal amplification and quantum dots for sensitive and photostable immunofluorescence detection. J Histochem Cytochem
2003; 51 (8): 981-7.
Hotz CZ. Applications of quantum dots in biology: an overview. In: Rosenthal SJ, Wright DW, editors. NanoBiotechnology Protocols. New Jersey: Humana Press; 2005. p. 1-17.
Smith AM, Nie S. Semiconductor nanocrystals: structure, properties, and band gap engineering. Acc Chem Res
2010; 43 (2): 190-200.
Bruchez M Jr., Moronne M, Gin P, Weiss S, Alivisatos AP. Semiconductor nanocrystals as fluorescent biological labels. Science
1998; 281 (5385): 2013-6.
Dubertret B, Skourides P, Norris DJ, Noireaux V, Brivanlou AH, Libchaber A. In vivo
imaging of quantum dots encapsulated in phospholipid micelles. Science
2002; 298 (5599): 1759-62.
Hardman R. A toxicologic review of quantum dots: toxicity depends on physicochemical and environmental factors. Environ Health Perspect
2006; 114 (2): 165-72.
Derfus AM, Chan WC, Bhatia SN. Probing the cytotoxicity of semiconductor quantum dots. Nano Lett
2004; 4 (1): 11-8.
Hezinger AF, Tessmar J, Göpferich A. Polymer coating of quantum dots - A powerful tool toward diagnostics and sensorics. Eur J Pharm Biopharm
2008; 68 (1): 138-52.
Lovriæ J, Cho SJ, Winnik FM, Maysinger D. Unmodified cadmium telluride quantum dots induce reactive oxygen species formation leading to multiple organelle damage and cell death. Chem Biol
2005; 12 (11): 1227-34.
Hoshino A, Fujioka K, Oku T, Suga M, Sasaki YF, Ohta T, Yasuhara M, Suzuki K, Yamamoto K. Physicochemical properties and cellular toxicity of nanocrystal quantum dots depend on their surface modification. Nano Lett
2004; 4 (11): 2163-9.
Hanaki K, Momo A, Oku T, Komoto A, Maenosono S, Yamaguchi Y, Yamamoto K. Semiconductor quantum dot/albumin complex is a long-life and highly photostable endosome marker. Biochem Biophys Res Commun
2003; 302 (3): 496-501.
Geys J, Nemmar A, Verbeken E, Smolders E, Ratoi M, Hoylaerts MF, Nemery B, Hoet PH. Acute toxicity and prothrombotic effects of quantum dots: impact of surface charge. Environ Health Perspect
2008; 116 (12): 1607-13.
Schipper ML, Iyer G, Koh AL, Cheng Z, Ebenstein Y, Aharoni A, Keren S, Bentolila LA, Li J, Rao J, Chen X, Banin U, Wu AM, Sinclair R, Weiss S, Gambhir SS. Particle size, surface coating, and PEGylation influence the biodistribution of quantum dots in living mice. Small
2009; 5 (1): 126-34.
Kelf TA, Sreenivasan VK, Sun J, Kim EJ, Goldys EM, Zvyagin AV. Non-specific cellular uptake of surface-functionalized quantum dots. Nanotechnology
2010; 21 (28): 285105.
Gao X, Yang L, Petros JA, Marshall FF, Simons JW, Nie S. In vivo
molecular and cellular imaging with quantum dots. Curr Opin Biotechnol
2005; 16 (1): 63-72.
Jaiswal JK, Mattoussi H, Mauro JM, Simon SM. Long-term multiple color imaging of live cells using quantum dot bioconjugates. Nat Biotechnol
2002; 21 (1): 47-51.
Lidke DS, Nagy P, Heintzmann R, Arndt-Jovin DJ, Post JN, Grecco HE, Jares-Erijman EA, Jovin TM. Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat Biotechnol
2004; 22 (2): 198-203.
Medintz IL, Clapp AR, Brunel FM, Tiefenbrunn T, Uyeda HT, Chang EL, Deschamps JR, Dawson PE, Mattoussi H. Proteolytic activity monitored by fluorescence resonance energy transfer through quantum-dot-peptide conjugates. Nat Mater
2006; 5 (7): 581-9.
Chan WC, Nie S. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science
1998; 281 (5385): 2016-8.
Pathak S, Davidson MC, Silva GA. Characterization of the functional binding properties of antibody conjugated quantum dots. Nano Lett
2007; 7 (7): 1839-45.
Goldman ER, Balighian ED, Mattoussi H, Kuno MK, Mauro JM, Tran PT, Anderson GP. Avidin: a natural bridge for quantum dot-antibody conjugates. J Am Chem Soc
2002; 124 (22): 6378-82.
Bruchez M Jr., Moronne M, Gin P, Weiss S, Alivisatos AP. Semiconductor nanocrystals as fluorescent biological labels. Science
1995; 281 (5385): 2013-6.
Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson JP, Ge N, Peale F, Bruchez MP. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat Biotechnol
2002; 21 (1): 41-6.
Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer A, McShane LM, Paik S, Pegram MD, Perez EA, Press MF, Rhodes A, Sturgeon C, Taube SE, Tubbs R, Vance GH, van de Vijver M, Wheeler TM, Hayes DF; American Society of Clinical Oncology; College of American Pathologists. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol
2006; 25 (1): 118-45.
Rojo MG, Bueno G, Slodkowska J. Review of imaging solutions for integrated quantitative immunohistochemistry in the pathology daily practice. Folia Histochem Cytobiol
2009; 47 (3): 349-54.
Wang HZ, Wang HY, Liang RQ, Ruan KC. Detection of tumor marker CA125 in ovarian carcinoma using quantum dots. Acta Biochim Biophys Sin (Shanghai)
2004; 36 (10): 681-6.
Colton HM, Falls JG, Ni H, Kwanyuen P, Creech D, McNeil E, Casey WM, Hamilton G, Cariello NF. Visualization and quantitation of peroxisomes using fluorescent nanocrystals: treatment of rats and monkeys with fibrates and detection in the liver. Toxicol Sci
2004; 80 (1): 183-92.
Wang J, Yong WH, Sun Y, Vernier PT, Koeffler HP, Gundersen MA, Marcu L. Receptor-targeted quantum dots: fluorescent probes for brain tumor diagnosis. J Biomed Opt
2007; 12 (4): 044021.
Cheng F, Yu W, Zhang X, Ruan Y. Quantum-dot-based technology for sensitive and stable detection of prostate stem cell antigen expression in human transitional cell carcinoma. Int J Biol Markers
2009; 24 (4): 271-6.
Petty HR, Elner VM, Kawaji T, Clark A, Thompson D, Yang DL. A facile method for immunofluorescence microscopy of highly autofluorescent human retinal sections using nanoparticles with large stokes shifts. J Neurosci Methods
2010; 191 (2): 222-6.
Qu YG, Zhang Q, Pan Q, Zhao XD, Huang YH, Chen FC, Chen HL. Quantum dots immunofluorescence histochemical detection of EGFR gene mutations in the non-small cell lung cancers using mutation-specific antibodies. Int J Nanomedicine
2014; 9: 5771-8.
Fountaine TJ, Wincovitch SM, Geho DH, Garfield SH, Pittaluga S. Multispectral imaging of clinically relevant cellular targets in tonsil and lymphoid tissue using semiconductor quantum dots. Mod Pathol
2006; 19 (9): 1181-91.
Wu A, True L, Gao X. Multiplexed and quantitative study of biomarker expression in tumor specimens using quantum dots. Proc SPIE
2006; (6096): 257-65.
Yezhelyev MV, Al-Hajj A, Morris C, Liu T, Lewis M, Cohen C, Zrazhevskiy P, Rogatko A, Nie S, Gao X. In situ
molecular profiling of breast cancer biomarkers with multicolor quantum dots. Adv Mater
2007; 19 (20): 3146-51.
Chaudry Q, Kong KY, Ahearn TU, Cohen V, Bostick RM, Wang MD. An Integrated Image Quantification System for Colorectal Cancer Risk Assessment Using Quantum Dots and Molecular Profiling. The 4 th
IEEE International Symposium on Biomedical Imaging: from Nano to Macro; 2007 April 12-15; Arlington, USA. Piscataway: IEEE; 2007. p. 1280-3.
Sweeney E, Ward TH, Gray N, Womack C, Jayson G, Hughes A, Dive C, Byers R. Quantitative multiplexed quantum dot immunohistochemistry. Biochem Biophys Res Commun
2008; 374 (2): 181-6.
Caldwell ML, Moffitt RA, Liu J, Parry RM, Sharma Y, Wang MD. Simple quantification of multiplexed quantum dot staining in clinical tissue samples. Conf Proc IEEE Eng Med Biol Soc
2008; 2008: 1907-10.
Chen H, Xue J, Zhang Y, Zhu X, Gao J, Yu B. Comparison of quantum dots immunofluorescence histochemistry and conventional immunohistochemistry for the detection of caveolin-1 and PCNA in the lung cancer tissue microarray. J Mol Histol
2009; 40 (4): 261-8.
Liu J, Lau SK, Varma VA, Moffitt RA, Caldwell M, Liu T, Young AN, Petros JA, Osunkoya AO, Krogstad T, Leyland-Jones B, Wang MD, Nie S. Molecular mapping of tumor heterogeneity on clinical tissue specimens with multiplexed quantum dots. ACS Nano
2010; 4 (5): 2755-65.
Liu J, Lau SK, Varma VA, Kairdolf BA, Nie S. Multiplexed detection and characterization of rare tumor cells in Hodgkin's lymphoma with multicolor quantum dots. Anal Chem
2010; 82 (14): 6237-43.
Chen C, Peng J, Xia H, Wu Q, Zeng L, Xu H, Tang H, Zhang Z, Zhu X, Pang D, Li Y. Quantum-dot-based immunofluorescent imaging of HER2 and ER provides new insights into breast cancer heterogeneity. Nanotechnology
2010; 21 (9): 095101.
Matsuno A, Itoh J, Takekoshi S, Nagashima T, Osamura RY. Three-dimensional imaging of the intracellular localization of growth hormone and prolactin and their mRNA using nanocrystal (quantum dot) and confocal laser scanning microscopy techniques. J Histochem Cytochem
2005; 53 (7): 833-8.
Orndorff RL, Warnement MR, Mason JN, Blakely RD, Rosenthal SJ. Quantum dot ex vivo
labeling of neuromuscular synapses. Nano Lett
2008; 8 (3): 780-5.
Sun JZ, Chen C, Jiang G, Tian WQ, Li Y, Sun SR. Quantum dot-based immunofluorescent imaging of Ki67 and identification of prognostic value in HER2-positive (non-luminal) breast cancer. Int J Nanomedicine
2014; 9: 1339-46.
Chen C, Peng J, Xia HS, Yang GF, Wu QS, Chen LD, Zeng LB, Zhang ZL, Pang DW, Li Y. Quantum dots-based immunofluorescence technology for the quantitative determination of HER2 expression in breast cancer. Biomaterials
2009; 30 (15): 2912-8.
Chen C, Sun SR, Gong YP, Qi CB, Peng CW, Yang XQ, Liu SP, Peng J, Zhu S, Hu MB, Pang DW, Li Y. Quantum dots-based molecular classification of breast cancer by quantitative spectroanalysis of hormone receptors and HER2. Biomaterials
2011; 32 (30): 7592-9.
Chen C, Xia HS, Gong YP, Peng J, Peng CW, Hu MB, Zhu XB, Pang DW, Sun SR, Li Y. The quantitative detection of total HER2 load by quantum dots and the identification of a new subtype of breast cancer with different 5-year prognosis. Biomaterials
2010; 31 (33): 8818-25.
Xue J, Chen H, Diao L, Chen X, Xia D. Expression of caveolin-1 in tongue squamous cell carcinoma by quantum dots. Eur J Histochem
2010; 54 (2): e20.
Chen SQ, Zhang HM, Li JB, Jiang HY, Fan L, Kong LZ, Yao SZ. Analyzing simultaneous positive expression of EZH2 and P53 protein to improve predictive value in cervical squamous cell carcinoma. Int J Gynecol Cancer
2014; 24 (9): 1653-8.
Korzhevskii DE, Kirik OV, Sukhorukova EG, Gilyarov AV. Use of semiconductor nanocrystals (quantum dots) in immunocytochemical studies. Neurosci Behav Physiol
2011; 41 (8): 799-802.
Yu J, Monaco SE, Onisko A, Bhargava R, Dabbs DJ, Cieply KM, Fine JL. A validation study of quantum dot multispectral imaging to evaluate hormone receptor status in ductal carcinoma in situ
of the breast. Hum Pathol
2013; 44 (3): 394-401.
Kairdolf BA, Mancini MC, Smith AM, Nie S. Minimizing nonspecific cellular binding of quantum dots with hydroxyl-derivatized surface coatings. Anal Chem
2008; 80 (8): 3029-34.
Barat B, Sirk SJ, McCabe KE, Li J, Lepin EJ, Remenyi R, Koh AL, Olafsen T, Gambhir SS, Weiss S, Wu AM. Cys-diabody quantum dot conjugates (immunoQdots) for cancer marker detection. Bioconjug Chem
2009; 20 (8): 1474-81.
Sukhanova A, Even-Desrumeaux K, Kisserli A, Tabary T, Reveil B, Millot JM, Chames P, Baty D, Artemyev M, Oleinikov V, Pluot M, Cohen JH, Nabiev I. Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes. Nanomedicine
2012; 8 (4): 516-25.
Li-Shishido S, Watanabe TM, Tada H, Higuchi H, Ohuchi N. Reduction in nonfluorescence state of quantum dots on an immunofluorescence staining. Biochem Biophys Res Commun
2006; 351 (1): 7-13.
Xiao Y, Gao X, Gannot G, Emmert-Buck MR, Srivastava S, Wagner PD, Amos MD, Barker PE. Quantitation of HER2 and telomerase biomarkers in solid tumors with IgY antibodies and nanocrystal detection. Int J Cancer
2008; 122 (10): 2178-86.
Zhou W, Schwartz DT, Baneyx F. Single-pot biofabrication of zinc sulfide immuno-quantum dots. J Am Chem Soc
2010; 132 (13): 4731-8.
Xing X, Zhang B, Wang X, Liu F, Shi D, Cheng Y. An "imaging-biopsy" strategy for colorectal tumor reconfirmation by multipurpose paramagnetic quantum dots. Biomaterials
2015; 48: 16-25.
Ferrara DE, Weiss D, Carnell PH, Vito RP, Vega D, Gao X, Nie S, Taylor WR. Quantitative 3D fluorescence technique for the analysis of en face preparations of arterial walls using quantum dot nanocrystals and two-photon excitation laser scanning microscopy. Am J Physiol Regul Integr Comp Physiol
2006; 290 (1): R114-23.
Killingsworth MC, Lai K, Wu X, Yong JL, Lee CS. Quantum dot immunocytochemical localization of somatostatin in somatostatinoma by widefield epifluorescence, super-resolution light, and immunoelectron microscopy. J Histochem Cytochem
2012; 60 (11): 832-43.
Rieger S, Kulkarni RP, Darcy D, Fraser SE, Köster RW. Quantum dots are powerful multipurpose vital labeling agents in zebrafish embryos. Dev Dyn
2005; 234 (3): 670-81.
Zahavy E, Freeman E, Lustig S, Keysary A, Yitzhaki S. Double labeling and simultaneous detection of B- and T-cells using fluorescent nano-crystal (q-dots) in paraffin-embedded tissues. J Fluoresc
2005; 15 (5): 661-5.
Au GH, Shih WY, Shih WH, Mejias L, Swami VK, Wasko K, Brooks AD. Assessing breast cancer margins ex vivo
using aqueous quantum-dot-molecular probes. Int J Surg Oncol
2012; 2012: 861257.
Hu M, Yan J, He Y, Lu H, Weng L, Song S, Fan C, Wang L. Ultrasensitive, multiplexed detection of cancer biomarkers directly in serum by using a quantum dot-based microfluidic protein chip. ACS Nano
2010; 4 (1): 488-94.
Tholouli E, Hoyland JA, Di Vizio D, O'Connell F, Macdermott SA, Twomey D, Levenson R, Yin JA, Golub TR, Loda M, Byers R. Imaging of multiple mRNA targets using quantum dot based in situ
hybridization and spectral deconvolution in clinical biopsies. Biochem Biophys Res Commun
2006; 348 (2): 628-36.
Naidoo K, Clay V, Hoyland JA, Swindell R, Linton K, Illidge T, Radford JA, Byers RJ. YY1 expression predicts favourable outcome in follicular lymphoma. J Clin Pathol
2011; 64 (2): 125-9.
Chen HL, Peng J, Zhu XB, Gao J, Xue JL, Wang MW, Xia HS. Detection of EBV in nasopharyngeal carcinoma by quantum dot fluorescent in situ
hybridization. Exp Mol Pathol
2010; 89 (3): 367-71.
Zhao Y, Ye M, Chao Q, Jia N, Ge Y, Shen H. Simultaneous detection of multifood-borne pathogenic bacteria based on functionalized quantum dots coupled with immunomagnetic separation in food samples. J Agric Food Chem
2009; 57 (2): 517-24.
[Figure 1], [Figure 2], [Figure 3]