Label-Free Multiplexed Microfluidic Analysis of Protein Interactions Based on Photonic Crystal Surface Mode Imaging.

High-throughput protein assays are crucial for modern diagnostics, drug discovery, proteomics, and other fields of biology and medicine. It allows simultaneous detection of hundreds of analytes and miniaturization of both fabrication and analytical procedures. Photonic crystal surface mode (PC SM) imaging is an effective alternative to surface plasmon resonance (SPR) imaging used in conventional gold-coated, label-free biosensors. PC SM imaging is advantageous as a quick, label-free, and reproducible technique for multiplexed analysis of biomolecular interactions. PC SM sensors are characterized by a longer signal propagation at the cost of a lower spatial resolution, which makes them more sensitive than classical SPR imaging sensors. We describe an approach for designing label-free protein biosensing assays employing PC SM imaging in the microfluidic mode. Label-free, real-time detection of PC SM imaging biosensors using two-dimensional imaging of binding events has been designed to study arrays of model proteins (antibodies, immunoglobulin G-binding proteins, serum proteins, and DNA repair proteins) at 96 points prepared by automated spotting. The data prove feasibility of simultaneous PC SM imaging of multiple protein interactions. The results pave the way to further develop PC SM imaging as an advanced label-free microfluidic assay for the multiplexed detection of protein interactions.

Enhancement of spontaneous emission of semiconductor quantum dots inside one-dimensional porous silicon photonic crystals.

Controlling spontaneous emission by modifying the local electromagnetic environment is of great interest for applications in optoelectronics, biosensing and energy harvesting. Although the development of devices based on one-dimensional porous silicon photonic crystals with embedded luminophores is a promising approach for applications, the efficiency of the embedded luminophores remains a key challenge because of the strong quenching of the emission due to the contact of the luminophores with the surface of porous silicon preventing the observation of interesting light-matter coupling effects. Here, we experimentally demonstrate an increase in the quantum dot (QD) spontaneous emission rate inside a porous silicon microcavity and almost an order of magnitude enhancement of QD photoluminescence intensity in the weak light-matter coupling regime. Furthermore, we have demonstrated drastic alteration of the QD spontaneous emission at the edge of the photonic band gap in porous silicon distributed Bragg reflectors and proved its dependence on the change in the density of photonic states.

Enhanced spontaneous emission from two-photon-pumped quantum dots in a porous silicon microcavity.

Photoluminescence (PL)-based sensing techniques have been significantly developed in practice due to their key advantages in terms of sensitivity and versatility of the approach. Recently, various nanostructured and hybrid materials have been used to improve the PL quantum yield and the spectral resolution. The near-infrared (NIR) fluorescence excitation has attracted much attention because it offers deep tissue penetration and it avoids the autofluorescence of the biological samples. In our study, we have shown both spectral and temporal PL modifications under two-photon excitation of quantum dots (QDs) placed in one-dimensional porous silicon photonic crystal (PhC) microcavities. We have demonstrated an up-to-4.3-fold Purcell enhancement of the radiative relaxation rate under two-photon excitation. The data show that the use of porous silicon PhC microcavities operating in the weak coupling regime permits the enhancement of the PL quantum yield of QDs under two-photon excitation, thus extending the limits of their biosensing applications in the NIR region of the optical spectrum.

Polariton-assisted splitting of broadband emission spectra of strongly coupled organic dye excitons in tunable optical microcavity.

Resonance interaction between a localized electromagnetic field and excited states in molecules paves the way to control fundamental properties of a matter. In this study, we encapsulated organic molecules with relatively low unoriented dipole moments in the polymer matrix, placed them in tunable optical microcavity and realized, for the first time, controllable modification of the broad photoluminescence (PL) emission of these molecules in strong coupling regime at room temperature. Notably, while in most previous studies it was reported that the single mode dominates in the PL signal (radiation of the so-called branch of the lower polariton), here we report on the observation of two distinct PL peaks, evolution of which has been followed as the microcavity mode is detuned from the excitonic resonance. A significant Rabi splitting estimated from the modified PL spectra was as large as 225 meV. The developed approach can be used both in fundamental research of resonant light-mater coupling and its practical applications in sensing and development of coherent spontaneous emission sources using a combination of carefully designed microcavity with a wide variety of organic molecules.

Engineering of Optically Encoded Microbeads with FRET-Free Spatially Separated Quantum-Dot Layers for Multiplexed Assays.

Quantum dot (QD) encoded microbeads are emerging for multiplexed analysis of biological markers. The quantitative encoding of microbeads prepared with different concentrations of QDs of different colors suffers from resonance energy transfer from the QDs fluorescing at shorter wavelengths to the QDs fluorescing at longer wavelengths. Here, we used the layer-by-layer deposition technique to spatially separate QDs of different colors with several polymer layers so that the distance between them would be larger than the Förster energy transfer radius. We performed fluorescence lifetime measurements to investigate and determine the conditions excluding significant resonance energy transfer between QDs within QD-encoded microbeads. Additionally, the number of QDs adsorbed onto microbeads was systematically established and multilayer structures of the QD-encoded microbead shells were characterized by scanning probe nanotomography. Finally, we prepared eight populations of FRET-free microbeads encoded with QDs of three colors at two intensity levels and demonstrated that all the optical codes are excitable at a single wavelength and may be clearly identified in three channels of a flow cytometer. The developed approach for engineering QD-encoded microbeads that are free from optical artefacts related to inter-QD resonance energy transfer paves the way to quantitative QD-based multiplexed assays.

Quantum Dot-Based Nanotools for Bioimaging, Diagnostics, and Drug Delivery.

Quantum dots (QDs) are highly fluorescent nanocrystals with advanced photophysical and spectral properties: high brightness and stability against photobleaching accompanied by broad excitation and narrow emission spectra. Water-soluble QDs functionalized with biomolecules, such as proteins, peptides, antibodies, and drugs, are used for biomedical applications. The advantages of QD-based approaches to immuno-histochemical analysis, single-molecule tracking, and in vivo imaging (over traditional methods with organic dyes and fluorescent proteins) are explained. The unique spectral properties of QDs offer opportunities for designing systems for multiplexed analysis by multicolor imaging for the simultaneous detection of multiple targets. Conjugation of drug molecules with QDs or their incorporation into QD-based drug-delivery particles makes it possible to monitor real-time drug tracking and carry out image-guided therapy. Because of the tunability of their photophysical properties, QDs emitting in the near-infrared have become an attractive tool for deep-tissue mono- and multiphoton in vivo imaging. We review recent achievements in QD applications for bioimaging, targeting, and drug delivery, as well as challenges related to their toxicity and non-biodegradability. Key and perspectives for further development of advanced QD-based nanotools are addressed.

Design, Synthesis, and Use of MMP-2 Inhibitor-Conjugated Quantum Dots in Functional Biochemical Assays.

The development of chemically designed matrix metalloprotease (MMP) inhibitors has advanced the understanding of the roles of MMPs in different diseases. Most MMP probes designed are fluorogenic substrates, often suffering from photo- and chemical instability and providing a fluorescence signal of moderate intensity, which is difficult to detect and analyze when dealing with crude biological samples. Here, an inhibitor that inhibits MMP-2 more selectively than Galardin has been synthesized and used for enzyme labeling and detection of the MMP-2 activity. A complete MMP-2 recognition complex consisting of a biotinylated MMP inhibitor tagged with the streptavidin-quantum dot (QD) conjugate has been prepared. This recognition complex, which is characterized by a narrow fluorescence emission spectrum, long fluorescence lifetime, and negligible photobleaching, has been demonstrated to specifically detect MMP-2 in in vitro sandwich-type biochemical assays with sensitivities orders of magnitude higher than those of the existing gold standards employing organic dyes. The approach developed can be used for specific in vitro visualization and testing of MMP-2 in cells and tissues with sensitivities significantly exceeding those of the best existing fluorogenic techniques.

Quantum dot surface chemistry and functionalization for cell targeting and imaging.

Quantum dots (QDs) are highly fluorescent nanoscale crystals with size-dependent emission spectra. Due to their excellent photophysical properties, QDs are a promising alternative to organic fluorescent dyes and fluorescent proteins for cell targeting, imaging, and drug delivery. For biomedical applications, QDs should be chemically modified to be stable in aqueous solutions and tagged with the recognition molecules or drugs. Here, we review surface modification approaches to, and strategies for, conjugation of bioactive molecules with QDs. There are a variety of methods of QD surface modification and QD incorporation into larger delivery systems that yield fluorescent nanocarriers from 10 nm to several micrometers. Conjugates of QDs with peptides, proteins, antibodies, oligonucleotides, and small molecules have been used for fluorescent targeting, tracking, and imaging both in vitro and in vivo. Due to an extremely high stability to photobleaching, QDs were used for long-term visualization. QD applications pave the way for new generations of ultrasensitive detection, diagnostic systems, as well as drug delivery approaches, combining accurate targeting, delivery, and imaging in a single assay.

Two-photon-induced Förster resonance energy transfer in a hybrid material engineered from quantum dots and bacteriorhodopsin.

Energy transfer from nanostructures to biological supramolecular photosystems is an important fundamental issue related to the possible influence of nanoobjects on biological functions. We demonstrate here two-photon-induced Förster resonance energy transfer (FRET) from fluorescent CdSe/ZnS quantum dots (QDs) to the photosensitive protein bacteriorhodopsin (bR) in a QD-bR hybrid material. The two-photon absorption cross section of QDs has been found to be about two orders of magnitude larger than that of bR. Therefore, highly selective two-photon excitation of QDs in QD-bR complexes is possible. Moreover, the efficiency of FRET from QDs to bR is sufficient to initiate bR photoconversion through two-photon excitation of QDs in the infrared spectral region. The data demonstrate that the effective spectral range in which the bR biological function is excited can be extended beyond the band where the protein itself utilizes light energy, which could open new ways to use this promising biotechnological material.

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates.

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.

Quantum dot-based lab-on-a-bead system for multiplexed detection of free and total prostate-specific antigens in clinical human serum samples.

An immunodiagnostic lab-on-a-bead suspension microarray based on microbeads encoded with quantum dots (QDs) has been developed and preclinically validated for multiplexed quantitative detection of prostate cancer markers in human serum samples. The sensitivity and specificity of the microarray are similar to those of "gold-standard" single-analyte ELISA. Moreover, the array has an improved immunoassay capacity, ensures quantitative detection of multiple cancer biomarkers and may be operational in a considerably wider dynamic range of concentrations. The array is characterized by reduced time and cost of analysis and is compatible with classical flow cytometers. Proof-of-concept preclinical tests ensured simultaneous quantitative determination of free and total prostate-specific antigens in human serum, with clear discrimination between the control and clinical samples. The proposed approach is flexible and paves the way to development of a wide variety of immunodiagnostic assays for multiplexed early diagnosis of various diseases. FROM THE CLINICAL EDITOR: Early diagnosis of cancer can result in better prognosis for patients. Thus, the use of specific tumor markers is widely employed in clinical practice. Traditional screening methods only employ single markers. The authors here developed a microarray system based on microbeads encoded with quantum dots (QDs), which can be used for multiplexed quantitative detection. The validated results on patient samples should lead to the development of a wider variety of assays for other diseases.

Multiphoton imaging of tumor biomarkers with conjugates of single-domain antibodies and quantum dots.

An ideal multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible two-photon absorption cross section (TPACS) and the smallest highly specific recognition molecules bound in an oriented manner. CdSe/ZnS quantum dots (QDs) conjugated to 13-kDa single-domain antibodies (sdAbs) derived from camelid IgG or streptavidin have been used as efficient two-photon excitation (TPE) probes for carcinoembryonic antigen (CEA) imaging on normal human appendix and colon carcinoma tissue. The TPACS for some conjugates was higher than 49,000 GM (Goeppert-Mayer units), considerably exceeding that of organic dyes being close to the theoretical value of 50,000 GM calculated for CdSe QDs. The ratio of sdAb-QD emission to the autofluorescence for 800 nm TPE was 40 times higher than that for 457.9 nm one-photon excitation. TPE ensures a clear discrimination of CEA-overexpressing tumor areas from normal tissue. Oriented sdAb-QD conjugates are bright specific labels for detecting low concentrations of antigens using multiphoton microscopy. FROM THE CLINICAL EDITOR: This study demonstrates carcinoembryonic antigen imaging on normal human appendix and colon carcinoma tissue utilizing CdSe/ZnS quantum dots conjugated to streptavidin or to 13-kDa single-domain antibodies as efficient two-photon excitation probes.

Cytotoxic Effects of Doxorubicin on Cancer Cells and Macrophages Depend Differently on the Microcarrier Structure.

Microparticles are versatile carriers for controlled drug delivery in personalized, targeted therapy of various diseases, including cancer. The tumor microenvironment contains different infiltrating cells, including immune cells, which can affect the efficacy of antitumor drugs. Here, prototype microparticle-based systems for the delivery of the antitumor drug doxorubicin (DOX) were developed, and their cytotoxic effects on human epidermoid carcinoma cells and macrophages derived from human leukemia monocytic cells were compared in vitro. DOX-containing calcium carbonate microparticles with or without a protective polyelectrolyte shell and polyelectrolyte microcapsules of about 2.4-2.5 µm in size were obtained through coprecipitation and spontaneous loading. All the microstructures exhibited a prolonged release of DOX. An estimation of the cytotoxicity of the DOX-containing microstructures showed that the encapsulation of DOX decreased its toxicity to macrophages and delayed the cytotoxic effect against tumor cells. The DOX-containing calcium carbonate microparticles with a protective polyelectrolyte shell were more toxic to the cancer cells than DOX-containing polyelectrolyte microcapsules, whereas, for the macrophages, the microcapsules were most toxic. It is concluded that DOX-containing core/shell microparticles with an eight-layer polyelectrolyte shell are optimal drug microcarriers due to their low toxicity to immune cells, even upon prolonged incubation, and strong delayed cytotoxicity against tumor cells.

Functionalized Calcium Carbonate-Based Microparticles as a Versatile Tool for Targeted Drug Delivery and Cancer Treatment.

Nano- and microparticles are increasingly widely used in biomedical research and applications, particularly as specific labels and targeted delivery vehicles. Silica has long been considered the best material for such vehicles, but it has some disadvantages limiting its potential, such as the proneness of silica-based carriers to spontaneous drug release. Calcium carbonate (CaCO3) is an emerging alternative, being an easily available, cost-effective, and biocompatible material with high porosity and surface reactivity, which makes it an attractive choice for targeted drug delivery. CaCO3 particles are used in this field in the form of either bare CaCO3 microbeads or core/shell microparticles representing polymer-coated CaCO3 cores. In addition, they serve as removable templates for obtaining hollow polymer microcapsules. Each of these types of particles has its specific advantages in terms of biomedical applications. CaCO3 microbeads are primarily used due to their capacity for carrying pharmaceutics, whereas core/shell systems ensure better protection of the drug-loaded core from the environment. Hollow polymer capsules are particularly attractive because they can encapsulate large amounts of pharmaceutical agents and can be so designed as to release their contents in the target site in response to specific stimuli. This review focuses first on the chemistry of the CaCO3 cores, core/shell microbeads, and polymer microcapsules. Then, systems using these structures for the delivery of therapeutic agents, including drugs, proteins, and DNA, are outlined. The results of the systematic analysis of available data are presented. They show that the encapsulation of various therapeutic agents in CaCO3-based microbeads or polymer microcapsules is a promising technique of drug delivery, especially in cancer therapy, enhancing drug bioavailability and specific targeting of cancer cells while reducing side effects. To date, research in CaCO3-based microparticles and polymer microcapsules assembled on CaCO3 templates has mainly dealt with their properties in vitro, whereas their in vivo behavior still remains poorly studied. However, the enormous potential of these highly biocompatible carriers for in vivo applications is undoubted. This last issue is addressed in depth in the Conclusions and Outlook sections of the review.

Photonic Crystal Surface Mode Real-Time Imaging of RAD51 DNA Repair Protein Interaction with the ssDNA Substrate.

Photonic crystals (PCs) are promising tools for label-free sensing in drug discovery screening, diagnostics, and analysis of ligand-receptor interactions. Imaging of PC surface modes has emerged as a novel approach to the detection of multiple binding events at the sensor surface. PC surface modification and decoration with recognition units yield an interface providing the highly sensitive detection of cancer biomarkers, antibodies, and oligonucleotides. The RAD51 protein plays a central role in DNA repair via the homologous recombination pathway. This recombinase is essential for the genome stability and its overexpression is often correlated with aggressive cancer. RAD51 is therefore a potential target in the therapeutic strategy for cancer. Here, we report the designing of a PC-based array sensor for real-time monitoring of oligonucleotide-RAD51 recruitment by means of surface mode imaging and validation of the concept of this approach. Our data demonstrate that the designed biosensor ensures the highly sensitive multiplexed analysis of association-dissociation events and detection of the biomarker of DNA damage using a microfluidic PC array. The obtained results highlight the potential of the developed technique for testing the functionality of candidate drugs, discovering new molecular targets and drug entities. This paves the way to further adaption and bioanalytical use of the biosensor for high-content screening to identify new DNA repair inhibitor drugs targeting the RAD51 nucleoprotein filament or to discover new molecular targets.

Basic principles and current trends in colloidal synthesis of highly luminescent semiconductor nanocrystals.

The principal methods for the synthesis of highly luminescent core-shell colloidal quantum dots (QDs) of the most widely used CdSe, CdS, ZnSe, and other A(II) B(VI) nanocrystals are reviewed. One-pot versus multistage core synthesis approaches are discussed. The noninjection one-pot method ensures slow, controllable growth of core nanocrystals starting from magic-size seed recrystallization, which yields defect-free cores with strictly specified sizes and shapes and a high monodispersity. Subsequent injection of shell precursors allows the formation of gradient core-shell QDs with a smooth potential barrier for electrons and holes, without strains or interfacial defects, and, as a consequence, a luminescence quantum yield (QY) approaching 100%. These general approaches can also be applied to semiconductor core-shell QDs other than A(II) B(VI) ones to cover the broad spectral range from the near-UV to IR regions of the optical spectrum, thus displacing fluorescent organic dyes from their application areas.

Biosensors Based on Inorganic Composite Fluorescent Hydrogels.

Fluorescent hydrogels are promising candidate materials for portable biosensors to be used in point-of-care diagnosis because (1) they have a greater capacity for binding organic molecules than immunochromatographic test systems, determined by the immobilization of affinity labels within the three-dimensional hydrogel structure; (2) fluorescent detection is more sensitive than the colorimetric detection of gold nanoparticles or stained latex microparticles; (3) the properties of the gel matrix can be finely tuned for better compatibility and detection of different analytes; and (4) hydrogel biosensors can be made to be reusable and suitable for studying dynamic processes in real time. Water-soluble fluorescent nanocrystals are widely used for in vitro and in vivo biological imaging due to their unique optical properties, and hydrogels based on these allow the preservation of these properties in bulk composite macrostructures. Here we review the techniques for obtaining analyte-sensitive fluorescent hydrogels based on nanocrystals, the main methods used for detecting the fluorescent signal changes, and the approaches to the formation of inorganic fluorescent hydrogels via sol-gel phase transition using surface ligands of the nanocrystals.

Nontoxic Fluorescent Nanoprobes for Multiplexed Detection and 3D Imaging of Tumor Markers in Breast Cancer.

Multiplexed fluorescent immunohistochemical analysis of breast cancer (BC) markers and high-resolution 3D immunofluorescence imaging of the tumor and its microenvironment not only facilitate making the disease prognosis and selecting effective anticancer therapy (including photodynamic therapy), but also provides information on signaling and metabolic mechanisms of carcinogenesis and helps in the search for new therapeutic targets and drugs. The characteristics of imaging nanoprobe efficiency, such as sensitivity, target affinity, depth of tissue penetration, and photostability, are determined by the properties of their components, fluorophores and capture molecules, and by the method of their conjugation. Regarding individual nanoprobe components, fluorescent nanocrystals (NCs) are widely used for optical imaging in vitro and in vivo, and single-domain antibodies (sdAbs) are well established as highly specific capture molecules in diagnostic and therapeutic applications. Moreover, the technologies of obtaining functionally active sdAb-NC conjugates with the highest possible avidity, with all sdAb molecules bound to the NC in a strictly oriented manner, provide 3D-imaging nanoprobes with strong comparative advantages. This review is aimed at highlighting the importance of an integrated approach to BC diagnosis, including the detection of biomarkers of the tumor and its microenvironment, as well as the need for their quantitative profiling and imaging of their mutual location, using advanced approaches to 3D detection in thick tissue sections. The existing approaches to 3D imaging of tumors and their microenvironment using fluorescent NCs are described, and the main comparative advantages and disadvantages of nontoxic fluorescent sdAb-NC conjugates as nanoprobes for multiplexed detection and 3D imaging of BC markers are discussed.

Impact of Macrophages on the Interaction of Cetuximab-Functionalized Polyelectrolyte Capsules with EGFR-Expressing Cancer Cells.

Polyelectrolyte capsules (PCs) are a promising tool for anticancer drug delivery and tumor targeting. Surface functionalization of PCs with antibodies is widely used for providing their specific interactions with cancer cells. The efficiency of PC-based targeted delivery systems can be affected by the cellular heterogeneity of the tumor, particularly by the presence of tumor-associated macrophages. We used human epidermoid carcinoma cells and macrophages derived from human leukemia monocytic cells in either monoculture or coculture to analyze the targeting capacity and internalization efficiency of PCs with a mean size of 1.03 ± 0.11 µm. The PCs were functionalized with the monoclonal antibody cetuximab targeting the human epidermal growth factor receptor (EGFR). We have shown that surface functionalization of the PCs with cetuximab ensures a specific interaction with EGFR-expressing cancer cells and promotes capsule internalization. In monoculture, the macrophages derived from human leukemia monocytic cells have been found to internalize both nonfunctionalized PCs and cetuximab-functionalized PCs (Cet-PCs) more intensely compared to epidermoid carcinoma cells. The internalization of Cet-PCs by cancer cells is mediated by lipid rafts of the cell membrane, whereas the PC internalization by macrophages is only slightly influenced by lipid rafts. Experiments with a coculture of human epidermoid carcinoma cells and macrophages derived from human leukemia monocytic cells have shown that Cet-PCs preferentially interact with cancer cells, which are subsequently attacked by macrophages. These data can be used to further improve the strategy of PC functionalization for targeted delivery, with the cellular heterogeneity of the tumor microenvironment taken into consideration.

Comparative efficiency of energy transfer from CdSe-ZnS quantum dots or nanorods to organic dye molecules.

Fluorescence resonance energy transfer (FRET) in conjugates of CdSe-ZnS semiconductor nanocrystals of different shapes (FRET donors) and an Alexa Fluor organic dye (FRET acceptors) is examined. The dye molecules are chemically conjugated with quantum dots (QDs) or nanorods (NRs) in dimethyl sulfoxide colloidal solutions, and FRET efficiency in the purified conjugates is measured. The FRET from NR to a single dye molecule is less efficient than that of the QD-dye conjugates and this effect is explained in terms of distance-limited energy-transfer rate in the case of a point-like acceptor and extended donor dipoles. However, the larger surface area of NRs allows for many more dye acceptors to be bound, and the total FRET efficiency in NR-dye conjugates approaches those of QD-dye conjugates.