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Ag2Te is one of the most promising semiconductors with a narrow band gap and low toxicity; however, it remains a challenge to tune the emission of Ag2Te quantum dots (QDs) precisely and continuously in a wide range. Herein, Ag2Te QDs emitting from 950 to 2100 nm have been synthesized via trialkylphosphine-controlled growth. Trialkylphosphine has been found to induce the dissolution of small-sized Ag2Te QDs due to its stronger ability to coordinate to the Ag ion than that of 1-octanethiol, predicated by the density functional theory. By controlling this dissolution effect, the monomer supply kinetics can be regulated, achieving precise size control of Ag2Te QDs. This synthetic strategy results in state-of-the-art silver-based QDs with emission tunability. Only by taking advantage of such an ultrawide emission has the sizing curve of Ag2Te been obtained. Moreover, the absolute photoluminescence quantum yield of Ag2Te QDs can reach 12.0% due to their well-passivated Ag-enriched surface with a density of 5.0 ligands/nm2, facilitating noninvasive in vivo fluorescence imaging. The high brightness in the long-wavelength near-infrared (NIR) region makes the cerebral vasculature and the tiny vessel with a width of only 60 μm clearly discriminable. This work reveals a nonclassical growth mechanism of Ag2Te QDs, providing new insight into precisely controlling the size and corresponding photoluminescence properties of semiconductor nanocrystals. The ultrasmall, low-toxicity, emission-tunable, and bright NIR-II Ag2Te QDs synthesized in this work offer a tremendous promise for multicolor and deep-tissue in vivo fluorescence imaging.

pH sensing plays a key role in the life sciences as well as the environmental, industrial, and agricultural fields. Carbon nanodots (C-dots) with small size, low toxicity, and excellent stability hold great potential in pH sensing as nanoprobes due to their intrinsic pH-sensitive photoluminescence (PL). Nonetheless, the undesirable sensitivity and response range of C-dot PL toward pH cannot meet the requirements of practical applications, and the unclear pH-sensitive PL mechanism makes it difficult to control their pH sensitivity. Herein, the quantitative correlation of pH-sensitive PL with specific surface structures of C-dots is uncovered for the first time, to our best knowledge. The association of carboxylate and H+ increases the ratio of nonradiation to radiation decay of C-dots through excited-state proton transfer, resulting in the decrease of PL intensity. Meanwhile, the dissociation of α-H in β-dicarbonyl forming enolate increases the extent of delocalization of the C-dots conjugated system, which induces the PL broadening to the red region and a decreasing intensity. Based on the understanding of the pH-sensitive PL mechanism, the pH-sensitive PL of C-dots can be switched by quantitative modulation of carboxyl and β-dicarbonyl groups to achieve a desirable pH response range with high sensitivity. This work contributes to a better understanding of the pH-sensitive PL of C-dots and therefore presents an effective strategy for controllably tuning their pH sensitivity, facilitating the rational design of C-dot-based pH sensors.

We proposed a method to regulate nucleic acid polymerization by proximity and designed an ultrasensitive biosensor based on proximity-induced exponential amplification reaction for proximity assay of proteins (streptavidin) and small molecules (adenosine triphosphate), which allows us to detect a variety of interesting targets by simply changing the binding sites of DNA.

We developed a purification strategy for amphiphilic polymer-coated QDs via salting out. Dynamic light scattering (DLS) results revealed that the zeta potential of octylamine-grafted poly-(acrylic acid) (OPA) micelles is closer to zero than that of OPA-coated QDs and can be compressed to electric neutrality first after adding NaCl, inducing the selective precipitation of OPA micelles.

Extracellular vesicles (EVs) are intercellular communicators that are heavily implicated in diverse pathological processes. However, it is poorly understood how EVs interact with recipient cells due to the lack of appropriate tracking techniques. Here, we report a robust chemoenzymatic labeling technique for visualizing the internalization process of EVs into target cells in real time. This method uses phospholipase D (PLD) to catalyze the in situ exchange of choline by alkyne in the native EV phosphatidylcholine. Subsequent alkyne-azide click chemistry allows conjugation of Cy5 dyes for visualizing EVs internalization by confocal fluorescence microscopy. The fluorescent labeling of EVs was accomplished in an efficient and biocompatible way, without affecting both the morphology and biological activity of EVs. We applied this chemoenzymatic labeling strategy to monitor the cellular uptake of cancer cell-derived EVs in real time and to further reveal multiple internalization mechanisms. This robust, biocompatible labeling strategy provides an essential tool for EV-related studies ranging from chemical biology to drug delivery.

Although plentiful advanced fluorescence sensors have achieved to analyze microRNAs (miRNAs) in living cells, the prerequisite relating to nucleic acids specific recognition based sensing principle compels them lack favorable accurancy and stability in such complicated biological mediums. Here, we make a double breakthrough for the two challenges by combining a near-infrared (NIR) light powering process with a DNA tetrahedron (DNAT)-based protection concept. In this sensing system, a special nanomachine is first engineered by conjugating a core-shell-structured upconversion nanoparticle capable of highly converting 808 nm NIR photons into ultraviolet ones with self-assembling DNATs. The newly developed nanostructure not only prevents the sensing pathway from triggering during the intracellular delivery as well as reducing the adverse thermal effect for cell viability but also significantly enhances the enzyme resistance to avoid degradation to produce false signals. Furthermore, a fluorescence resonance energy transfer sensing strategy is rationally designed on this nanomachine. Upon using the powering light to excite the upconversion luminescence to activate the nanomachine in living cells, it can stably trace the precise level changes of miRNA-21 sequences at the reaching position with an "off-on" mode of fluorescence outputs.

Different kinds of artificial drug delivery systems (DDS) have been widely exploited and utilized toward effective tumor therapy. Establishing a biocompatible DDS with a flexible release of the therapeutic agents has been a challenges and an impetus to the development of tumor diagnosis and therapy fields. Herein, the chemotherapeutic agents doxorubicin hydrochloride (DOX) and photosensitizer indocyanine green (ICG) were simultaneously packaged into the cavity of microvesicles (MVs) through the electroporation technique. With the aid of MVs-based DDS, the packaged therapeutic agents could be effectively delivered into the target tumor cells. The ambient temperature sharply increased because of controllable external near-infrared (NIR) laser irradiation, which induced the cracking of MVs-based DDS, directly accompanied by the dynamic and controllable release of DOX and ICG. Almost all the tumor cells could be killed by the synergistic effect of the released DOX and ICG. This research successfully established a smart DDS with NIR laser inducing controllable release of therapeutic agents for effective synergistic photochemotherapy toward cervical cancer.

Abnormal expression of microRNAs is closely related to human diseases. Ultrasensitive detection of microRNAs with wide linear dynamic range is necessary as the concentrations of microRNAs distributed in biological samples usually range from fM to nM. Here, we constructed an ultrasensitive electrochemical sensor for microRNA-21 based on the integration of a dual signal amplification strategy of hybridization chain reaction (HCR) and enzyme-induced metallization (EIM) with anodic stripping voltammetry (ASV). MicroRNA-21 was captured by capture probe H1 (CP H1) modified magnetic nanobeads (MBs) and amplified by HCR. Multiple alkaline phosphatases (ALP) were coupled based on the specific reaction of biotin and streptavidin. A dual amplification strategy of HCR and EIM enhanced electrochemical signal by approximately 120-fold from the perspectives of target amplification and detection signal amplification, which decreased the detection limit of microRNA-21 to 0.12 fM. Compared to the reports previously reported, a wider linear dynamic range of 2.5 fM to 25 nM spanning 7 orders of magnitude was obtained. The method provides high sensitivity, specificity and a wider linear dynamic range, which shows a great potential in the early diagnosis of diseases.

We report a new upconversion nanoparticles (UCNPs) based luminescent resonance energy transfer (LRET) analytical platform by making use of optical tweezers technology. The LRET model is designed by simultaneously conjugating Yb3+ and Er3+ co-doped UCNPs (as the donors) and tetramethyl rhodamine (TAMRA) molecules (as the acceptors) on microspheres to fabricate complex microspheres. Upon a single complex microsphere entering the three-dimensional potential well formed with a tightly focused 980 nm Guassian-shaped laser beam, it is optically trapped and concurrently the upconversion emission is excited, whereby the donor signals are transferred to the acceptors. As a proof-of-concept investigation, microRNA-21 sequences are selected as the targets, by which the distance between the two perfectly matched luminophors is controlled to several nanometers via nucleic acid hybridization. Without the involvement of luminescence amplification strategies, the proposed single microsphere based LRET method shows highly competitive sensitivity with a limit of detection down to 114 fM and satisfactory specificity towards microRNAs detection. Moreover, its practical working ability is demonstrated by credibly quantifying the absolute contents of miRNA-21 sequences in three cancer cell lines and even tracing the targets in as few as 100 cancer cells. Thus, this favorable analytical methodology provides an alternative for bioassays and holds certain potential in biomedical applications.

Unlike other extracellular vesicle (EV) subtypes such as exosomes, the lack of well-defined universal markers on the surface of microvesicles (MVs) has led to difficulty in the detection of the entire MV population. To design a universal MV detection method, we reported highly sensitive electrical detection of MVs using a reduced graphene oxide (RGO)-based field-effect transistor (FET) biosensor by the introduction of a membrane biotinylation strategy in this work. Biotinylated MVs (B-MVs) were obtained by supplying the culture medium with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG-biotin) while cultivating the cells. Excellent biotinylation efficiency of MVs (92.6%) was then realized. A streptavidin (SA) probe was subsequently modified onto the channel surface of the as-fabricated RGO-based FET device, which was capable of specifically recognizing B-MVs due to the high affinity between SA and biotin in a 1 : 4 recognition format. The results showed that the RGO-based FET biosensor could detect B-MVs in a wide range from 105 particles per mL to 109 particles per mL with a low detection limit down to 20 particles per μL, which was the lowest value compared with other previously reported results. This platform also allowed distinguishing B-MVs from other unbiotinylated EV types such as MVs and exosomes, exhibiting excellent specificity. Moreover, this FET biosensor demonstrated the capability of detecting B-MVs derived from different cell lines including cancer cells and normal cells, indicating its versatility and potential applications in the biomedical field.

Fluorescent nanoparticles exhibit unique optical properties for applications in imaging and sensing. Herein, the enhancement of luminescence emission from self-assembled nanoparticles including gold nanoclusters, cadmium sulphide and near-infrared silver selenide quantum dots are reported. Addition of zinc and pyrophosphate ions induces assembly and disassembly of the particle aggregates and the corresponding intensified and recovery of fluorescence intensity, respectively. The enhancement is due to assembly-enhanced emission of metal nanoclusters and passivation of the surface trap states of quantum dots. With the hydrolysis of pyrophosphate catalyzed by alkaline phosphatase, the released zinc ions from pyrophosphate-zinc ion complexes mediate nanoparticle assembly and lead to luminescence regeneration. The assembly-induced fluorescence enhancement can be applied for sensitive assay of alkaline phosphatase with a detection limit of 3.4 pM in a continuous and real-time way. This method is capable of screening enzyme inhibitor and analyzing biological samples.

We incorporate three conceptual components including luminescence-concentrating upconversion nanoparticles, optical tweezers, and DNA walkers into bead carriers to establish a new imaging analysis.

Quantum dots (QDs) possess optical properties of superbright fluorescence, excellent photostability, narrow emission spectra, and optional colors. Labeled with QDs, single molecules/viruses can be rapidly and continuously imaged for a long time, providing more detailed information than labeled with other fluorophores. While they are widely used to label proteins in single-molecule tracking studies, QDs have rarely been used to study virus infection, mainly due to lack of accepted labeling strategies. Here, we report a general method to mildly and readily label enveloped viruses with QDs. Lipid-biotin conjugates were used to recognize and mark viral lipid membranes, and streptavidin (SA)-QD conjugates were used to light them up. Such a method allowed enveloped viruses to be labeled in 2 hours with specificity and efficiency up to 99% and 98%. The intact morphology and the native infectivity of viruses could be furthest preserved. With the aid of this QD labeling method, we lit wild-type (WT) and mutant Japanese encephalitis virus (JEV) up, tracked their infection in living Vero cells, and found that H144A and Q258A substitutions in the envelope (E) protein didn’t affect the virus intracellular trafficking. The lipid-specific QD labeling method described in this study provides a handy and practical tool to readily “see” the viruses and follow their infection, facilitating the widespread use of single-virus tracking and the uncovering of complex infection mechanisms.Author summaryVirus infection in host cells is a complex process comprising a large number of dynamic molecular events. Single-virus tracking is a versatile technique to study these events. To perform this technique, viruses must be fluorescently labeled to be visible to fluorescence microscopes. Quantum dot is a kind of fluorescent tags that has many unique optical properties. It has been widely used to label proteins in single-molecule tracking studies, but rarely used to study virus infection, mainly due to lack of accepted labeling method. In this study, we developed a lipid-specific method to readily, mildly, specifically, and efficiently label enveloped viruses with quantum dots by recognizing viral envelope lipids with lipid-biotin conjugates and recognizing these lipid-biotin conjugates with streptavidin-quantum dot conjugates. Such a method is superior to the commonly used DiD/DiO labeling and the other QD labeling methods. It is not only applicable to normal viruses, but also competent to label the key protein-mutated viruses and the inactivated high virulent viruses, providing a powerful tool for single-virus tracking.

Although suspension bead-based assay technology has been widely used owing to its advantages of high-throughput and microvolume detection, its sensitivity is greatly limited because it detects the fluorescence signal emitted by microbeads for a short time in the flowing fluid. In this work, we present the approach for prostate-specific antigen (PSA) detection of both free PSA (fPSA) and total PSA (tPSA) based on bead-array based fluorescence imaging by combining multiple optical trapping and bead-based bioassays. The polystyrene beads were employed to enrich the targets using the classic sandwich immuno-binding and tagged with fluorescent quantum dots (QDs), and the QDs-tagged beads in suspension were trapped array-by-array using multiple optical tweezers constructed with a diffraction optical element and excited with a 405 nm fiber laser for wide-field fluorescence imaging. The distinctive size information from the image of the trapped beads enabled the sorting of different targets. Moreover, the limits of detection for fPSA and tPSA are 3.8 pg/mL and 2.5 pg/mL respectively with good specificity. More importantly, this strategy was successfully used to detect fPSA and tPSA simultaneously in real serum samples. The high sensitivity, good selectivity, and tiny sample volume make this strategy a promising method for life sciences and clinical applications.

With suppressed photon scattering and diminished autofluorescence, in vivo fluorescence imaging in the 1,500- to 1,700-nm range of the near-IR (NIR) spectrum (NIR-IIb window) can afford high clarity and deep tissue penetration. However, there has been a lack of NIR-IIb fluorescent probes with sufficient brightness and aqueous stability. Here, we present a bright fluorescent probe emitting at ∼1,600 nm based on core/shell lead sulfide/cadmium sulfide (CdS) quantum dots (CSQDs) synthesized in organic phase. The CdS shell plays a critical role of protecting the lead sulfide (PbS) core from oxidation and retaining its bright fluorescence through the process of amphiphilic polymer coating and transferring to water needed for imparting aqueous stability and compatibility. The resulting CSQDs with a branched PEG outer layer exhibited a long blood circulation half-life of 7 hours and enabled through-skin, real-time imaging of blood flows in mouse vasculatures at an unprecedented 60 frames per second (fps) speed by detecting ∼1,600-nm fluorescence under 808-nm excitation. It also allowed through-skin in vivo confocal 3D imaging of tumor vasculatures in mice with an imaging depth of ∼1.2 mm. The PEG-CSQDs accumulated in tumor effectively through the enhanced permeation and retention effect, affording a high tumor-to-normal tissue ratio up to ∼32 owing to the bright ∼1,600-nm emission and nearly zero autofluorescence background resulting from a large ∼800-nm Stoke's shift. The aqueous-compatible CSQDs are excreted through the biliary pathway without causing obvious toxicity effects, suggesting a useful class of ∼1,600-nm emitting probes for biomedical research.

Currently, the biosynthesis of nanomaterials by organisms is attracting considerable attention because of the sustainable and environmentally friendly nature of the reactions involved in this process compared with those in the conventional nanomaterial synthesis. However, the manipulation and control of nanomaterial biosynthesis remain difficult because of the lack of knowledge about the biosynthetic mechanisms. In the present study, we elucidated the selenium (Se)-precursor and Se metabolic flux in the biosynthesis of cadmium-selenium quantum dots (CdSe QDs) in Saccharomyces cerevisiae and improved the cells’ ability to biosynthesize CdSe QDs through gene modification based on the regulation mechanism. By deleting the genes involved in Se metabolism and measuring seleno-amino acids, we identified selenocysteine (SeCys) as the primary Se-precursor in the intracellular biosynthesis of CdSe QDs. Further studies demonstrated that the selenomethionine (SeMet)-to-SeCys pathway regulates CdSe QD biosynthesis. Knowledge of the regulatory pathway allowed us to enhance SeMet synthesis by overexpression of the MET6 gene, and an increased CdSe QD yield was realized in the engineered cells. Understanding the mechanism of CdSe QD biosynthesis helped to determine the relationship between nanocrystal formation and biological processes, and offers a new perspective to manipulation of nanomaterial biosynthesis.

Mechanofluorochromic materials, which change their photoluminescence (PL) colors in responding to mechanical stimuli, can be used as mechanosensors, security papers, and photoelectronic devices. However, traditional mechanofluorochromic materials can only be adjusted to a monotone direction upon the external stimuli. Controllable pressure-triggered blue- and red-shifted PL is reported for C-dots. The origin of mechanofluorochromism (MFC) in C-dots is interpreted based on structure-property relationships. The carbonyl group and the π-conjugated system play key roles in the PL change of C-dots under high pressure. As the pressure increases, the enhanced π-π stacking of the π-conjugated system causes the red-shift of PL, while the conversion of carbonyl groups eventually induces a blue-shift. Together with their low toxicity, good hydrophilicity, and small size, the tunable MFC property would boost various potential applications of C-dots.

A facile and controllable method for one-step synthesis of small-sized monodisperse fluorescent-magnetic bifunctional microparticles based on droplet splitting and photopolymerization was established. Using this method, magnetic nanoparticles and acryloyl rhodamine B (acryloyl-RhB) molecules were incorporated into the dispersed phase, which allowed the direct generation of functional microparticles. Functional microparticles with diameters in the range of 2.8 to 19.2 μm were obtained by controlling the fluidic characteristics. The fluorescence intensity of the microparticles could be regulated by adjusting the concentration of the acryloyl-RhB molecule in the dispersed phase. Furthermore, the fluorescent-magnetic bifunctional microparticles were successfully used to detect proteins specifically by ratiometric fluorescence analysis, showing their potential in biological sample detection.

All-inorganic CsPbX3 (X = I, Br, Cl) perovskite nanocrystals have emerged as a new class of optoelectronic material with tunable emission wavelengths, narrow full-width at half-maximum (FWHM) and high photoluminescence quantum yields (PLQYs) (≥50% in general). However, the instability caused by high delocalization of surface ions is still the major issue that obstructs their application. The type of surface ligands plays a key role in the stability of perovskite nanocrystals. In this work, stable cubic Br-rich CsPbBr3 quantum dots (QDs) with uniform particle sizes, high quantum yields and high crystallinity were obtained by a one-step hot-injection synthesis method. Oleylamine (OM) as one of the ligands in the classic synthesis method was replaced with (3-aminopropyl)triethoxysilane (APTES). The stability of the CsPbBr3 QDs was significantly improved under different testing conditions.

Amyotrophic lateral sclerosis (ALS) involves the abnormal posttranslational modifications and fibrillization of copper, zinc superoxide dismutase (SOD1) and TDP-43. However, how SOD1-catalyzed reaction product hydrogen peroxide affects amyloid formation of SOD1 and TDP-43 remains elusory. 90% of ALS cases are sporadic and the remaining cases are familial ALS. In this paper, we demonstrate that H2O2 at pathological concentrations triggers the fibrillization of wild-type SOD1 both in vitro and in SH-SY5Y cells. Using an anti-dimedone antibody that detects sulfenic acid modification of proteins, we found that Cys-111 in wild-type SOD1 is oxidized to C-SOH by pathological concentration of H2O2, followed by the formation of sulfenic acid modified SOD1 oligomers. Furthermore, we show that such SOD1 oligomers propagate in a prion-like manner, and not only drive wild-type SOD1 to form fibrils in the cytoplasm but also induce cytoplasm mislocalization and the subsequent fibrillization of wild-type TDP-43, thereby inducing apoptosis of living cells. Thus, we propose that H2O2 at pathological concentrations triggers the fibrillization of wild-type SOD1 and subsequently induces SOD1 toxicity and TDP-43 toxicity in neuronal cells via sulfenic acid modification of Cys-111 in SOD1. Our Western blot and ELISA data demonstrate that sulfenic acid modified wild-type SOD1 level in cerebrospinal fluid of 15 sporadic ALS patients is significantly increased compared with 6 age-matched control patients. These findings can explain how H2O2 at pathologic concentrations regulates the misfolding and toxicity of SOD1 and TDP-43 associated with ALS, and suggest that sulfenic acid modification of wild-type SOD1 should play pivotal roles in the pathogenesis of sporadic ALS.

Due to a growing trend in their biomedical application, biosynthesized nanomaterials are of great interest to researchers nowadays with their biocompatible, low-energy consumption, economic, and tunable characteristics. It is important to understand the mechanism of biosynthesis in order to achieve more efficient applications. Since there are only rare studies on the influences of cellular energy levels on biosynthesis, the influence of energy is often overlooked. Through determination of the intracellular ATP concentrations during the biosynthesis process, significant changes were observed. In addition, ATP synthesis deficiency caused great decreases in quantum dots (QDs) biosynthesis in the Δatp1, Δatp2, Δatp14, and Δatp17 strains. With inductively coupled plasma-atomic emission spectrometry and atomic absorption spectroscopy analyses, it was found that ATP affected the accumulation of the seleno-precursor and helped with the uptake of Cd and the formation of QDs. We successfully enhanced the fluorescence intensity 1.5 or 2 times through genetic modification to increase ATP or SeAM (the seleno analog of S-adenosylmethionine, the product that would accumulate when ATP is accrued). This work explains the mechanism for the correlation of the cellular energy level and QDs biosynthesis in living cells, demonstrates control of the biosynthesis using this mechanism, and thus provides a new manipulation strategy for the biosynthesis of other nanomaterials to widen their applications.

Cell-derived microvesicles (MVs), which are biogenic nanosized membrane-bound vesicles that convey bioactive molecules between cells, have recently received attention for use as natural therapeutic platforms. However, the medical applications of MV-based delivery platforms are limited by the lack of effective methods for the efficient isolation of MVs and the convenient tuning of their targeting properties. Herein, we report the development of magnetic and folate (FA)-modified MVs based on a donor cell-assisted membrane modification strategy. MVs inherit the membrane properties of their donor cells, which allows them to be modified with the biotin and FA on their own membrane. By conjugating with streptavidin-modified iron oxide nanoparticles (SA-IONPs), the MVs can be conveniently, efficiently, and rapidly isolated from the supernatant of their donor cells using magnetic activated sorting. Moreover, the conjugated magnetic nanoparticles and FA confer magnetic and ligand targeting activities on the MVs. Then, the MVs were transformed into antitumor delivery platforms by directly loading doxorubicin via electroporation. The modified MVs exhibited significantly enhanced antitumor efficacy both in vitro and in vivo. Taken together, this study provides an efficient and convenient strategy for the simultaneous isolation of cell-derived MVs and transformation into targeted drug delivery nanovectors, thus facilitating the development of natural therapeutic nanoplatforms.

Atherosclerosis (AS) is a major contributor to cardiovascular diseases worldwide, and alleviating inflammation is a promising strategy for AS treatment. Here, we report molecularly engineered M2 macrophage-derived exosomes (M2 Exo) with inflammation-tropism and anti-inflammatory capabilities for AS imaging and therapy. M2 Exo are derived from M2 macrophages and further electroporated with FDA-approved hexyl 5-aminolevulinate hydrochloride (HAL). After systematic administration, the engineered M2 Exo exhibit excellent inflammation-tropism and anti-inflammation effects via the surface-bonded chemokine receptors and the anti-inflammatory cytokines released from the anti-inflammatory M2 macrophages. Moreover, the encapsulated HAL can undergo intrinsic biosynthesis and metabolism of heme to generate anti-inflammatory carbon monoxide and bilirubin, which further enhance the anti-inflammation effects and finally alleviate AS. Meanwhile, the intermediate protoporphyrin IX (PpIX) of the heme biosynthesis pathway permits the fluorescence imaging and tracking of AS. This work was supported by the National Natural Science Foundation of China (No. 91859123 and 21874011), the National Science and Technology Major Project (No. 2018ZX 10301405-001), and China Postdoctoral Science Foundation (No. 2018M630076).

The authors describe a field effect transistor (FET) based immunoassay for the detection of inactivated ebola virus (EBOV). An equine antibody against the EBOV glycoprotein was immobilized on the surface of the FET that was previously modified with reduced graphene oxide (RGO). The antibody against EBOV was immobilized on the modified FET, and the response to EBOV was measured as a function of the shift of Dirac voltage. The method can detect the EBOV over the concentration range from 2.4 × 10−12 g·mL−1 to 1.2 × 10−7 g·mL−1 and with a limit of detection as low as 2.4 pg·mL−1. The assay has satisfactory specificity and was applied to the quantitation of inactivated EBOV in spiked serum.

Fluorescence anisotropy (FA) has been widely applied for detecting and monitoring special targets in life sciences. However, matrix autofluorescence restricted its further application in complex biological samples. Herein, we report a near-infrared-II (NIR-II) FA strategy for detecting adenosine triphosphate (ATP) in human serum samples and breast cancer cell lysate, which employed NIR-II fluorescent Ag2Se quantum dots (QDs) as tags to reduce matrix autofluorescence effect and applied graphene oxide (GO) to enhance fluorescence anisotropy signals. In the presence of ATP, the recognition between NIR-II Ag2Se QDs labeled aptamer (QD-pDNA) and ATP led to the release of QD-pDNA from GO, resulting in the obvious decrease of FA values. ATP could be quantitatively detected in concentrations ranged from 3 nM to 2500 nM, with a detection limit down to 1.01 nM. This study showed that the developed NIR-II FA strategy could be applied for detecting targets in complex biological samples and had great potential for monitoring interactions between biomolecules in biomedical research.

The accurate and rapid determination of hydrogen peroxide (H2O2) levels is of great significance because of the far-ranging impacts of H2O2 homeostasis on human health. In this work, we developed an enhanced electrochemiluminescence (ECL) biosensor based on CdZnSeS quantum dots (QDs) condensed with ordered mesoporous carbon (OMC) for high-sensitive detection of H2O2 in live cells. Benefiting from the combination of QDs with the highly conductive OMC materials, the prepared ECL biosensor exhibited outstanding ECL signal. We then assessed the analytical performance of the ECL biosensor for H2O2 detection, and found that the biosensor possessed low detection limit, wide linear range, desirable selectivity, and long-time stability. By the detection of H2O2 in different live cells, the ECL biosensor platform shows its potential for reliable and sensitive H2O2 detection in diagnostic applications and physiological research.

Real-time and in-situ monitoring of H2S in biological system has become crucial but faces big challenge since H2S was identified as an important endogenous gas signaling molecule. Herein we prepare a poly(3,4-ethylenedioxythiophene) (PEDOT) modified nanoporous gold (NPG) microelectrode for electrochemical detection of H2S. NPG framework possesses excellent conductivity, 3D porous structure and large specific area. PEDOT sensing film effectively retards the electrode fouling during detection, which ensures a stable response towards H2S over the range of interest. The synergetic properties of NPG scaffold and PEDOT sensing films endow the microelectrode with high sensitivity and fast response, thus allowing monitoring of H2S release from single cells. This work represents the first step toward real-time electrochemical monitoring of H2S from single cells, therefore potentially facilitate future studies to better understand the function of H2S in biological and pathological processes.

We developed a fluorescent aptasensor based on the making use of double-stranded DNA (dsDNA)/graphene oxide (GO) as the signal probe and the activities of exonuclease I (Exo I). This method takes advantage of the stronger affinity of the aptamer to its target rather than to its complementary sequence (competitor), and the different interaction intensity of dsDNA, mononucleotides with GO. Specifically, in the absence of target, the competitor hybridizes with the aptamer, preventing the digestion of the competitor by Exo I, and thus the formed dsDNA is adsorbed on GO surface, allowing fluorescence quenching. When the target is introduced, the aptamer preferentially binds with its target. Thereby, the corresponding nuclease reaction takes place, and slight fluorescence change is obtained after the introduction of GO due to the weak affinity of the generated mononucleotides to GO. Adenosine (AD) was chosen as a model system and tested in detail. Under the optimized conditions, smaller dissociation constant (Kd, 311.0 µM) and lower detection limit (LOD, 3.1 µM) were obtained in contrast with traditional dye-labeled aptamer/GO based platform (Kd=688.8 µM, LOD=21.2 µM). Satisfying results were still obtained in the evaluation of the specificity and the detection of AD in human serum, making it a promising tool for the diagnosis of AD-relevant diseases. Moreover, we demonstrated the effect of the competitor on the LOD, and the results reveal that the sensitivity could be enhanced by using the rational competitor. The present design not only constructs a label-free aptamer based platform but also extends the application of dsDNA/GO complex in biochemical and biomedical studies.

Bifunctional magnetic nanobeads (bi-MBs) were fabricated by co-immobilizing target recognition molecules and signal molecules on a magnetic nanobead surface, which were used as both separation and enrichment carriers and signal carriers. The bi-MBs could capture and separate avian influenza A (H7N9) virus (H7N9 AIV) from complex samples efficiently based on the specific reaction between antigen–antibody and their good magnetic response, which simplified sample pretreatment and saved the detection time. Taking advantages of their high surface to volume ratio and rich surface functional groups, multiple alkaline phosphatase (ALP) signal molecules were tethered on the surface of bi-MBs which greatly amplified the detection signal. As an efficient signal amplification strategy, enzyme-induced metallization had been integrated with bi-MBs and anodic stripping voltammetry to construct an ultrasensitive electrochemical immunosensor for H7N9 AIV detection. Under the optimal conditions, the introduction of bi-MBs could amplify the detection signal in about four times compared with the same immunoassay without MBs, and the method showed a wide linear range of 0.01–20 ng/mL with a detection limit of 6.8 pg/mL. The electrochemical immunosensor provides a simple and reliable platform with high sensitivity and selectivity which shows great potential in early diagnosis of diseases.

A method of fluorescent nanoparticle-based indirect immunofluorescence assay using either fluorescence microscopy or flow cytometry for the rapid detection of pathogenic Escherichia coli O157:H7 was developed. The dye-doped silica nanoparticles (NPs) were synthesized using W/O microemulsion methods with the combination of 3-aminopropyltriethoxysilane (APTES) and fluorescein isothiocyanate (FITC) and polymerization reaction with carboxyethylsilanetriol sodium salt (CEOS). Protein A was immobilized at the surface of the NPs by covalent binding to the carboxyl linkers and the surface coverage of Protein A on NPs was determined by the Bradford method. Rabbit anti-E. Coli O157:H7 antibody was used as primary antibody to recognize E. coli O157:H7 and then antibody binding protein (Protein A) labeled with FITC-doped silica NPs (FSiNPs) was used to generate fluorescent signal. With this method, E. Coli O157:H7 in buffer and bacterial mixture was detected. In addition, E. coli O157:H7 in several spiked background beef samples were measured with satisfactory results. Therefore, the FSiNPs are applicable in signal-amplified bioassay of pathogens due to their excellent capabilities such as brighter fluorescence and higher photostability than the direct use of conventional fluorescent dyes.

Metal chalcogenide semiconductor nanocrystals (NCs) are ideal inorganic materials for solar cells and biomedical labeling. In consideration of the hazard and instability of alkylphosphines, the phosphine-free synthetic route has become one of the most important trends in synthesizing selenide QDs. Here we report a novel phase transfer strategy to prepare phosphine-free chalcogenide precursors. The anions in aqueous solution were transferred to toluene via electrostatic interactions between the anions and didodecyldimethylammonium bromide (DDAB). The obtained chalcogenide precursors show high reactivity with metal ions in the organic phase and could be applied to the low-temperature synthesis of various metal chalcogenide NCs based on a simple reaction between metal ions (e.g. Ag(+), Pb(2+), Cd(2+)) and chalcogenide anions (e.g. S(2-)) in toluene. In addition to chalcogenide anions, other anions such as BH4(-) ions and AuCl4(-) ions can also be transferred to the organic phase for synthesizing noble metal NCs (such as Ag and Au NCs).

A novel fluorescence “turn-on” strategy, which is based on the formation of Hg2+-mediated molecular-beacons (MBs), the preferable cleavage capacity of exonuclease III to double-stranded DNA compared with single-stranded one, and the remarkable difference in the binding ability of graphene oxide (GO) with single-stranded DNA and the mononucleotides, is designed for Hg2+ assay. The Hg2+-mediated base pairs facilitate the dye labeled MBs to fold into a hairpin structure, which is more likely to be digested by exonuclease III, and an obvious increase in the fluorescence intensity is observed after incubating with GO due to the weak affinity of the product-mononucleotides to GO. A fluorescent “turn-on” method based on graphene oxide and exonuclease III was designed for Hg2+ assay. The introduction of GO greatly increases the signal-to-background ratio, and the sensitivity is significantly improved due to the amplified capability of exonuclease III. Under the optimal conditions, Hg2+ is specifically and sensitively detected with a detection limit of 0.83 nM. Compared with the reported Hg2+ assay methods, the proposed strategy is simple, cost effective and selective, which might provide a new platform for developing a sensitive Hg2+ biosensor. Mercury level in the blood is an important indicator of mercury poisoning in clinical study. To testify the possibility of the use of this method for the assay of Hg2+ in real samples, detection of Hg2+ in 1% human serum was investigated and satisfactory results were obtained, which suggests that this method has great potential for bioanalysis.

A green room-temperature method based on phase transfer and a reaction between Zn2+ and S2− in toluene for coating a ZnS shell on CdSe and Ag2S quantum dots (QDs), respectively, has been developed. In this method, the S2− anions in Na2S aqueous solution were transferred to toluene via electrostatic interactions between the anions and didodecyldimethylammonium bromide (DDAB), which served as an excellent sulfur precursor and thereby reacted with Zn2+ to form a ZnS shell on the surface of semiconductor QDs in the presence of QDs in an organic phase. After the ZnS shell was coated on QDs, the photoluminescence (PL) intensities of the CdSe and Ag2S QDs were found to increase approximately 12 times and 14 times, respectively, while both the emission wavelengths and the peak shapes were well maintained. The proposed method is novel, simple and robust, providing a green solution to the preparation of ZnS shell-coated QDs.

Membrane nanotubes (MNTs) are physical connections for intercellular communication and induced by various viruses. However, the formation of vaccinia virus (VACV)-induced MNTs has never been studied. In this report, VACV-induced MNTs formation process was monitored on a microfluidic chip equipped with a series of side chambers, which protected MNTs from fluidic shear stress. MNTs were formed between susceptible cells and be facilitated by VACV infection through three patterns. The formed MNTs varied with cell migration and virus concentration. The length of MNTs was positively correlated with the distance of cell migration. With increasing virus titer, the peak value of the ratio of MNT-carried cell appeared earlier. The immunofluorescence assay indicated that the rearrangement of actin fibers induced by VACV infection may lead to the formation of MNTs. This study presents evidence for the formation of MNTs induced by virus and helps us to understand the relationship between pathogens and MNTs.

We present a simple and sensitive method for multiplexed DNA detection by simultaneously capturing two different DNA sequences with a same silica nanoparticle (NP) through a sandwich mode. This biobarcode assay method was demonstrated by using oligonucleotide sequences of 64 bases associated with human papillomavirus (HPV) 16 and 18 L1 genes as model systems. The nonfluorescent carboxyl-modified silica NPs were prepared using water-in-oil (W/O) microemulsion methods. Avidin was immobilized on the surface of the NPs by covalent binding to the carboxyl linkers. The binding capacity of the avidin-covered NPs for ligand biotin was quantified and the results show that about 8 avidin molecules are bound to one nanoparticle. The silica nano-platforms were prepared through the biotin–avidin interaction and the amounts of capture DNA strands for HPV-16 and HPV-18 (C-16 and C-18, respectively) conjugated to the surface of the same NPs were measured using fluorescent dye hoechst33258. The calculated result shows that the amounts of conjugated C-16 and C-18 on 1 mg of NPs (9.2 pmol) are about 13.5 pmol and 15.5 pmol, respectively. A one-step hybridization reaction was performed by mixing the silica nano-platforms, HPV-16 and HPV-18 target DNA (T-16 and T-18), fluorescein amidite (FAM) or 6-carboxyl-X-rhodamine (Rox) labeled HPV-16 and HPV-18 probes. The hybrid-conjugated NPs were separated by centrifugation, and T-16 and T-18 were detected by measuring fluorescence signals of FAM and Rox respectively. The results show linear dependence of the fluorescence intensity on target DNA concentration in the range from 0.5 to 9 nM, and the detection limit (3 σ ) of T-16 and T-18 is 0.17 nM and 0.78 nM, respectively.

Cell-to-cell spread of virus is a comprehensive process with involvement of cellular actin cytoskeleton and substrate topography can affect the arrangement of cytoskeleton via contact guidance, yet interaction among virus, cytoskeleton and substrate topography is still unknown. To investigate the virus-cell-substrate interaction, we designed a microgrooved poly(dimethyl siloxane) (PDMS) substrate for the study of vaccinia virus (VACV) cell-to-cell spread and the remodeling of cellular actin cytoskeleton in viral infection process. Interestingly, VACV-induced plaques on microgrooved substrate were elliptical instead of circular plaques on smooth substrate, suggesting an anisotropic cell-to-cell spread of VACV. The spread rate was faster in the direction parallel to microgroove and slower in the direction perpendicular to microgroove than that on smooth substrate. Host cells cultured on microgrooved surface showed significant alignment and elongation in the axis parallel to microgrooves. Cell elongation is one reason for anisotropic spread but could not totally explain the phenomenon. Actin fibers in infected cells maintained alignment and VACV-induced actin tails tipped with virions were oriented along the direction parallel to microgroove. These results suggested that substrate topography can affect infected cells and these effects will guide the spread of virus via orientation of actin cytoskeleton. This work opens a window for understanding virus response to substrate topography, and has potential implications on revealing virus-cell-substrate interactions in vivo.

Two different functional groups modified RuBpy-doped silica fluorescent nanoprobes Probe A and B that conjugated with avidin were prepared for the recognition of liver cancer cells. Firstly RuBpy-doped silica nanoparticles were synthesized by reverse microemulsion and then modified with different functional groups. Probe A was prepared by the conjugation of avidin with carboxyl modified nanoparticles through covalent binding using EDC/sulfo-NHS, whereas Probe B was prepared by the conjugation of avidin with the PEG linkers on the surface nanoparticles using cyanogen bromide method. Therefore, different from Probe A, Probe B was obtained by coupling avidin to the nanoparticles through long-chain PEG molecules. The two probes were incubated with liver cancer cells respectively, and microscopic fluorescence imaging showed that Probe B which contained PEG molecules was more effectively applied for the recognition of tumor marker carcinoembryonic antigen (CEA) in liver cancer cells.

The study on circulating tumor cells (CTCs) has great significance for cancer prognosis, treatment monitoring, and metastasis diagnosis, in which isolation and enrichment of CTCs are key steps due to their extremely low concentration in peripheral blood. Herein, magnetic nanospheres (MNs) were fabricated by a convenient and highly controllable layer-by-layer assembly method. The MNs were nanosized with fast magnetic response, and nearly all of the MNs could be captured by 1 min attraction with a commercial magnetic scaffold. In addition, the MNs were very stable without aggregation or precipitation in whole blood and could be re-collected nearly at 100% in a monodisperse state. Modified with anti-epithelial-cell-adhesion-molecule (EpCAM) antibody, the obtained immunomagnetic nanospheres (IMNs) successfully captured extremely rare tumor cells in whole blood with an efficiency of more than 94% via only a 5 min incubation. Moreover, the isolated cells remained viable at 90.5 ± 1.2%, and they could be directly used for culture, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry (ICC) identification. ICC identification and enumeration of the tumor cells in the same blood samples showed high sensitivity and good reproducibility. Furthermore, the IMNs were successfully applied to the isolation and detection of CTCs in cancer patient peripheral blood samples, and even one CTC in the whole blood sample was able to be detected, which suggested they would be a promising tool for CTC enrichment and detection.

In this work, the cellular uptake, elimination and toxicity of CdSe/ZnS QDs in HepG2 cells were comprehensively studied using inductively coupled plasma mass spectrometry (ICP-MS), MTT assay, AO/EB staining, and glutathione level and gene expression analysis. ICP-MS analytical results showed that the uptake efficiency of CdSe QDs by HepG2 cells was lower than that of Cd(II) and Se(IV), and the uptake was dose- and time-dependent. The uptake amount was related to the physicochemical properties of QDs, and NH2-QDs with smaller size were more easily taken up by cells. In combination with various biochemical methodologies, a systematic and thorough quantitative analysis of the in vitro effects of CdSe/ZnS QDs with different coatings was conducted, along with that of Cd (II) and Se (IV). Although Cd(II) above 8.9 μM exhibited obvious toxicity to the cells, no obvious toxicity of four CdSe/ZnS QDs was observed within the tested concentration range (10-100 nM), most likely due to the protection of the ZnS shell and the PEG coating. From the molecular level's point of view, QDs at concentration of 100 nM exhibit obvious impact on the cells, such as increased gene expression (MT1A and CYP1A1), which was positively correlated with the intracellular concentration of QDs.

QD biosynthesis affects the mechanical strength of yeast cells. The intracellular synthesis of CdSe QD in yeast cells incubated with Na2 SeO3 and subsequently with CdCl2 increases the glucan content of their cell walls, resulting in their enhanced mechanical strength.

A sandwich immunoassay method for rapid detection of dual cancer biomarkers in serum on a magnetic field controllable microfluidic chip (MFCM-Chip) was established. A nickel pattern was used to generate high magnetic field gradients to increase the magnetic force on the superparamagnetic beads (SPMBs), which enabled the rapid generation of controllable SPMB patterns in microfluidic channels. The SPMB patterns could keep stable during the fast continuous washing process even at a flow rate of 50 μL/min. This approach demonstrated excellent specificity because the fast continuous washing could remove non-specifically adsorptive contaminants more efficiently than fixed volume batch washing. This approach was used to simultaneously detect carcinoma embryonic antigen (CEA) and α-fetoprotein (AFP) directly in serums. The whole on-chip detection was finished within 40 min, which was much faster than conventional enzyme-linked immunosorbent assay (ELISA) method. High luminescent streptavidin modified QDs (SA-QDs) were used as fluorescence indicators, and the detection limits were 3.5 ng/mL and 3.9 ng/mL for CEA and AFP, respectively. The linear ranges were from 10.0 ng/mL to 800.0 ng/mL. With the high sensitivity, high selectivity and short assay time, this approach could be used for rapid, high throughput detection of cancer biomarkers in clinical trials.

Microbial cells have shown a great potential to biosynthesize inorganic nanoparticles within their orderly regulated intracellular environment. However, very little is known about the mechanism of nanoparticle biosynthesis. Therefore, it is difficult to control intracellular synthesis through the manipulation of biological processes. Here, we present a mechanism-oriented strategy for controlling the biosynthesis of fluorescent CdSe quantum dots (QDs) by means of metabolic engineering in yeast cells. Using genetic techniques, we demonstrated that the glutathione metabolic pathway controls the intracellular CdSe QD formation. Inspired from this mechanism, the controllability of CdSe QD yield was realized through engineering the glutathione metabolism in genetically modified yeast cells. The yeast cells were homogeneously transformed into more efficient cell-factories at the single-cell level, providing a specific way to direct the cellular metabolism toward CdSe QD formation. This work could provide the foundation for the future development of nanomaterial biosynthesis.

An ultrasensitive and highly efficient assay for real-time monitoring of nitric oxide (NO) at single-cell level based on a reduced graphene oxide (RGO) and iron-porphyrin-functionalized graphene (FGPCs) field-effect transistor (FET) biosensor is reported. A layer-to-layer assembly of RGO and FGPCs on a prefabricated FET sensor surface through π-π stacking interaction allowed superior electrical conductivity caused by RGO, and highly catalytic specificity induced by metalloporphyrin, ensuring the ultrasensitive and highly specific detection of NO. The results demonstrated that the RGO/FGPCs FET biosensor was capable of real-time monitoring of NO in the range from 1 pM to 100 nM with the limit of detection as low as 1 pM in phosphate-buffered saline (PBS) and 10 pM in the cell medium, respectively. Moreover, the developed biosensor could be used for real-time monitoring of NO released from human umbilical vein endothelial cells (HUVECs) at single-cell level. Along with its miniaturized sizes, ultrasensitive characteristics, and fast response, the FET biosensor is promising as a new platform for potential biological and diagnostic applications.

Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity.

Due to excellent optical properties, quantum dots (QDs) have been widely applied to sensing, labeling, and imaging. For the fabrication of QD-based bioprobes, purification is usually the crucial step. Hydrophilic octylamine grafted polyacrylic acid modified QDs (OPA-QDs) were prepared, and purified by high-performance size exclusion chromatography (HPSEC) to remove excess OPA and aggregated QDs. The percentage of suspended agglomerates of OPA-QDs in the unpurified OPA-QDs increases from 4% to 31% through a year, but the purified OPA-QDs of the same batch possess excellent colloidal stability for at least one year. Subsequently, QD-based bioprobes were fabricated by the conjugation between QDs and streptavidin (SA) or antibody (IgG), generating QD-SA and QD-IgG, respectively, which were purified via HPSEC. Finally, the resulting QD-SA and QD-IgG were adopted to detect tumour markers on slices and showed specific positive signals without nonspecific adsorption, which was contrary to the unpurified QD-IgG. Thus, the HPSEC-coupled system proposed in the current work is potent and universal for the generation of purified and monodisperse QD-based bioprobes, which is promising in the nanobiodetection field.

Weikun Tang,1 Junpeng Fan,1 Yide He,1 Bihai Huang,2 Huihui Liu,1 Daiwen Pang,2 Zhixiong Xie11College of Life Sciences, 2College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, People's Republic of ChinaAbstract: Quantum dots (QDs) have many potential clinical and biological applications because of their advantages over traditional fluorescent dyes. However, the genotoxicity potential of QDs still remains unclear. In this paper, a plasmid-based system was designed to explore the genotoxic mechanism of QDs by detecting changes in DNA configuration and biological activities. The direct chemicobiological interactions between DNA and mercaptoacetic acid-coated CdSe-core QDs (MAA–QDs) were investigated. After incubation with different concentrations of MAA–QDs (0.043, 0.13, 0.4, 1.2, and 3.6 µmol/L) in the dark, the DNA conversion of the covalently closed circular (CCC) DNA to the open circular (OC) DNA was significantly enhanced (from 13.9% ± 2.2% to 59.9% ± 12.8%) while the residual transformation activity of plasmid DNA was greatly decreased (from 80.7% ± 12.8% to 13.6% ± 0.8%), which indicated that the damages to the DNA structure and biological activities induced by MAA–QDs were concentration-dependent. The electrospray ionization mass spectrometry data suggested that the observed genotoxicity might be correlated with the cadmium–mercaptoacetic acid complex (Cd–MAA) that is formed in the solution of MAA–QDs. Circular dichroism spectroscopy and transformation assay results indicated that the Cd–MAA complex might interact with DNA through the groove-binding mode and prefer binding to DNA fragments with high adenine and thymine content. Furthermore, the plasmid transformation assay could be used as an effective method to evaluate the genotoxicities of nanoparticles.Keywords: genotoxicity, MAA CdSe quantum dots, cadmium–MAA complex, transformation assay, DNA&nbsp

In this work, robust approach for a highly sensitive point-of-care virus detection was established based on immunomagnetic nanobeads and fluorescent quantum dots (QDs). Taking advantage of immunomagnetic nanobeads functionalized with the monoclonal antibody (mAb) to the surface protein hemagglutinin (HA) of avian influenza virus (AIV) H9N2 subtype, H9N2 viruses were efficiently captured through antibody affinity binding, without pretreatment of samples. The capture kinetics could be fitted well with a first-order bimolecular reaction with a high capturing rate constant k(f) of 4.25 × 10(9) (mol/L)(-1) s(-1), which suggested that the viruses could be quickly captured by the well-dispersed and comparable-size immunomagnetic nanobeads. In order to improve the sensitivity, high-luminance QDs conjugated with streptavidin (QDs-SA) were introduced to this assay through the high affinity biotin-streptavidin system by using the biotinylated mAb in an immuno sandwich mode. We ensured the selective binding of QDs-SA to the available biotin-sites on biotinylated mAb and optimized the conditions to reduce the nonspecific adsorption of QDs-SA to get a limit of detection low up to 60 copies of viruses in 200 μL. This approach is robust for application at the point-of-care due to its very good specificity, precision, and reproducibility with an intra-assay variability of 1.35% and an interassay variability of 3.0%, as well as its high selectivity also demonstrated by analysis of synthetic biological samples with mashed tissues and feces. Moreover, this method has been validated through a double-blind trial with 30 throat swab samples with a coincidence of 96.7% with the expected results.

Labeling of virus opens new pathways for the understanding of viruses themselves and facilitates the utilization of viruses in modern biology, medicine, and materials. Based on the characteristic that viruses hijack their host cellular machineries to survive and reproduce themselves, a host-cell-assisted strategy is proposed to label enveloped viruses. By simply feeding Vero cells with commercial 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (Biotin-Cap-PE), we obtained biotinylated Vero cells whose membrane systems were modified with biotin. Subsequently, pseudorabies viruses (PrV) were cultivated in the biotinylated Vero cells, and the PrV progenies were spontaneously labeled with Biotin-Cap-PE during viral natural assembly process. Since the viral natural assembly process was employed for the labeling, potential threats of genetic engineering and difficulties in keeping viral natural bioactivity were avoided. Importantly, this labeling strategy for enveloped virus greatly reduces the technical complexity and allows researchers from different backgrounds to apply it for their specified demands.

Ultrabright carbon nanodots-hybridized silica nanospheres (CSNs) are synthesized through the Stober process of silane functionalized C-dots. The fluorescence of carbon nanodots is converged intensely. A CSN is about 3800 times brighter than a single-carbon nanodot. Along with their high brightness and low cytotoxicity, CSNs also indicate their potential application in cellular labeling.

A novel molecular aptamer beacon (MAB) was designed by integrating a single-labeled hairpin-shaped aptamer and graphene oxide (GO). The hairpin-shaped aptamer was constructed with anti-ATP aptamer and another five nucleotides added to the 5'-end of the aptamer which are complementary to nucleotides at the 3'-end of the aptamer to form a hairpin-shaped probe. This newly designed MAB which acts as a low background signal platform was used for the ATP detection based on long-range resonance energy transfer (LrRET). In the absence of ATP, the adsorption of the dye-labeled hairpin-shaped aptamer on GO makes the dyes close proximity to GO surface resulting in high efficiency quenching of fluorescence of the dyes. Therefore, the fluorescence of the designed MAB is completely quenched by GO, and the system shows very low background. Conversely, and very importantly, upon the adding of ATP, the quenched fluorescence is recovered significantly, and ATP can be detected in a wide range of 5-2500μM with a detection limit of 2μM and good selectivity. Moreover, when the GO-based MAB was used in cellular ATP assays, preeminent fluorescence signals were obtained, thus the platform of GO-based MAB could be used to detect ATP in real-world samples.