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Fluorescence imaging in the second near-infrared (NIR-II, 1000-1700 nm) region holds great promise for in vivo bioimaging. However, it is challenging to develop a brilliant donor-acceptor-donor (D-A-D) type NIR-II fluorophore with maximal absorption beyond 1000 nm in aqueous solution. Herein, we report a bright D-A-D type BOIMPY-based NIR-II dye (NK1143) with peak absorption/emission at 1005/1143 nm for in vivo bioimaging. Co-assembly of NK1143, SC12 (intermolecular steric hindrance modulator), and DSPE-PEG2000 effectively inhibits H-aggregation of NK1143 in aqueous solution and enhances the brightness simultaneously up to 53-fold by leveraging synergistic steric regulation strategy. Notably, this strategy allows for deep optical penetration of 8 mm and high-resolution blood vessels imaging in vivo, displaying high signal-to-background ratio of 7.8/1 under 980 nm excitation. More importantly, the BOIMPY-based nanoprobe can passively target and clearly visualize broad types of tumor xenografts, further improving intraoperative NIR-II fluorescence-guided resection of tiny metastases of less than 1 mm. This work provides an effective strategy for the development of BOIMPY-based NIR-II organic fluorophores with broad applications.

Bacterial infectious diseases are common clinical diseases that seriously threaten human health, especially in countries and regions with poor environmental hygiene. Due to the lack of characteristic clinical symptoms and signs, it is a challenge to distinguish a bacterial infection from other infections, leading to misdiagnosis and antibiotic overuse. Therefore, there is an urgent need to develop a specific method for detection of bacterial infections. Herein, utilizing ultrabright fluorescent nanospheres (FNs) as reporters, immunochromatographic dyad test strips are developed for the early detection of bacterial infections and distinction of different stages of bacterial infectious diseases in clinical samples. C-reactive protein (CRP) and heparin-binding protein (HBP) are quantified and assayed because their levels in plasma are varied dynamically and asynchronously during the progression of the disease. The detection limits of CRP and HBP can reach as low as 0.51 and 0.65 ng/mL, respectively, due to the superior fluorescence intensity of each FN, which is 570 times stronger than that of a single quantum dot. The assay procedure can be achieved in 22 min, fully meeting the needs of rapid and ultrasensitive detection in the field. This constructed strip has been successfully used to profile the stage and severity of bacterial infections by monitoring the levels of CRP and HBP in human plasma samples, showing great potential as a point-of-care biosensor for clinical diagnosis. In addition to bacterial infections, the developed ultrabright FN-based point-of-care testing can be readily expanded for rapid, quantitative, and ultrasensitive detection of other trace substances in complex systems.

Generally speaking, it is difficult to keep nanomaterials encapsulated in amphiphilic polymers like octylamine-grafted poly(acrylic acid) (OPA) compact in coating-layer, with a small hydrodynamic size. Here, we prepared stable hydrophilic quantum dots (QDs) via encapsulation in ∼3 nm-long amphiphilic and zwitterionic (AZ) molecules. After encapsulation with AZ molecules, the coated QDs are only 2.1 nm thicker in coating, instead of 5.4 nm with OPA. Meanwhile, the hydrodynamic sizes of CdSe/CdS, ZnCdSeS, ZnCdSe/ZnS, and CdSe/ZnS QDs encapsulated in AZ molecules (AZ-QDs) are less than 15 nm, and 6-7 nm smaller than those of QDs in OPA (OPA-QDs). Notably, both extracellular and intracellular nonspecific binding of AZ-QDs is approximately 100-folds lower than that of OPA-QDs.

Mammalian cell display technology uses eukaryotic protein expression system to display proteins on cell surfaces and has become an important method in biological research. Although mammalian cell display technology has many advantages and development potential, certain attributes of the displayed protein remain uncharacterized, such as whether the displayed proteins re-enter the cell and how displayed proteins move into the cell. Here, we present the endocytosis mechanism, motility behavior, and transport kinetics of displayed proteins determined using HaloTag as the displayed protein and quantum dot-based single-particle tracking. The displayed protein enters the cell through clathrin-mediated endocytosis and is transported through the cell in three stages, which is dependent on microfilaments and microtubules. The dynamic information obtained in this study provides answers to questions about endocytosis and postendocytosis transport of displayed proteins and, therefore, is expected to facilitate the development of surface display technology.

Despite that the currently discovered CRISPR-Cas12a system is beneficial for improving the detection accuracy and design flexibility of luminescent biosensors, there are still challenges to extend target species and strengthen adaptability in complicated biological media. To conquer these obstacles, we present here some useful strategies. For the former, the limitation to nucleic acids assay is broken through by introducing a simple functional DNA regulation pathway to activate the unique trans-cleavage effect of this CRISPR system, under which the expected biosensors are capable of effectively transducing a protein (employing dual aptamers) and a metal ion (employing DNAzyme). For the latter, a time-gated luminescence resonance energy transfer imaging manner using a long-persistent nanophosphor as the energy donor is performed to completely eliminate the background interference and a nature-inspired biomimetic periodic chip constructed by photonic crystals is further combined to enhance the persistent luminescence. In line with the above efforts, the improved CRISPR-Cas12a luminescent biosensor not only exhibits a sound analysis performance toward the model targets (carcinoembryonic antigen and Na+) but also owns a strong anti-interference feature to actualize accurate sensing in human plasma samples, offering a new and applicative analytical tool for laboratory medicine.

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.

Microtubules (MTs) are the main component of cytoskeletons, providing long tracks for cargo trafficking across the cytoplasm. In the past years, transport along MTs was frequently reported to be rapid directed motions with speeds of several micrometers per second, but is that all the truth? Using single-particle tracking, we roundly and precisely analyzed the dynamic behaviors of three kinds of cargoes transported along MTs in two types of cells. It was found that during the transport processes, the directed motions of the cargoes were frequently interrupted by nondirected motions which greatly reduced the translocation rate toward the nucleus. What is more, in addition to the widely reported rapid directed motions, a type of directed motions with most speeds below 0.5 μm/s occurred more frequently. On the whole, these slow directed motions took longer than the rapid directed motions and resulted in displacements same as those of the rapid ones. To sum up, while travelling along MTs toward the cell interior, endocytosed cargoes moved alternately in rapid-directed, slow-directed and nondirected modes. In this process, the rapid- and the slow-directed motions contributed almost equally to the cargoes' translocation. This work provides original insights into the transport on MTs, facilitating a more comprehensive understanding of intracellular trafficking.

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.

Taking advantage of outstanding precision in target recognition and trans-cleavage ability, the recently discovered CRISPR/Cas12a system provides an alternative opportunity for designing fluorescence biosensors. To fully exploit the analytical potential, we introduce here some meaningful concepts. First, the collateral cleavage of CRISPR/Cas12a is efficiently activated in a functional DNA regulation manner and the bottleneck which largely applicable to nucleic acids detection is broken. After selection of a representative aptamer and DNAzyme as the transduction pathways, the sensing coverage is extended to a small organic compound (ATP) and a metal ion (Na+). The assay sensitivity is significantly improved by utilizing a bead-supported enrichment strategy wherein emerging holographic optical tweezers are used to enhance imaging stability and simultaneously achieve multiflux analysis. Last, a sandwich-structured energy-concentrating upconversion nanoparticle triggered boosting luminescent resonance energy transfer mode is comined to face with complicated biological samples by skillfully confining the emitters into a very limited inner shell. Following the above attempts, the developed CRISPR/Cas12a biosensors not only present an ultrasensitive assay behavior toward these model non-nucleic acid analytes but also can serve as a formidable toolbox for determining real samples including single cell lysates and human plasma, proving a good practical application capacity.

Influenza A virus (IAV) is internalized into its host cells by endocytosis, which involves many cellular proteins and molecules. In this study, we focus on the function of calcium ion (Ca2+) in IAV...

An ultrasensitive electrochemiluminescence (ECL) biosensor was proposed based on a closed bipolar electrode (BPE) for the detection of alkaline phosphatase (ALP). For most of the BPE-ECL biosensors, an effective signal amplification strategy was the key to enhance the sensitivity of the system. Herein, the signal amplification strategy of the enzyme catalysis was utilized in the BPE-ECL system. Au nanoparticles (NPs) were electrodeposited on the cathode surface of the ITO electrode to improve the stability and sensitivity of the signal. Compared with the previous BPE-ECL biosensors, the sensitivity was increased by at least 3 orders of magnitude. The biosensor showed high sensitivity and specificity of ALP detection with a detection limit of as low as 3.7 aM. Besides, it was further applied to the detection of ALP in different types of cells and successfully realized ALP detection in single Hep G2 cell, which had a huge application prospect in single biomolecule detection or single cell analysis.

Multifunctional nanoparticles hold great potential for clinical and medical research. Herein, Ag2Te QDs were applied as multifunctional contrast agents for the second near-infrared region (NIR-II) ...

Uncovering the mechanisms of virus infection and assembly is crucial for preventing the spread of viruses and treating viral disease. The technique of single-virus tracking (SVT), also known as single-virus tracing, allows one to follow individual viruses at different parts of their life cycle and thereby provides dynamic insights into fundamental processes of viruses occurring in live cells. SVT is typically based on fluorescence imaging and reveals insights into previously unreported infection mechanisms. In this review article, we provide the readers a broad overview of the SVT technique. We first summarize recent advances in SVT, from the choice of fluorescent labels and labeling strategies to imaging implementation and analytical methodologies. We then describe representative applications in detail to elucidate how SVT serves as a valuable tool in virological research. Finally, we present our perspectives regarding the future possibilities and challenges of SVT.

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.

We demonstrate an effective approach to realize active and real-time temperature monitoring around the gold nanobipyramids (AuNBPs)-labeled cancer cell under 808 nm laser irradiation by combining optical tweezers and temperature-sensitive upconversion microparticles (UCMPs). On the one hand, the aptamer-modified AuNBPs that absorb laser at 808 nm not only act as an excellent photothermal reagent but also accurately and specifically bind the target cancer cells. On the other hand, the single optically trapped NaYF4:Yb3+, Er3+ UCMPs with a 980 nm laser exhibit temperature-dependent luminescence properties, where the intensity ratio of emission 525 and 547 nm varies with the ambient temperature. Therefore, real-time temperature variation monitoring is performed by 3D manipulation of the trapped single UCMP to control its distance from the AuNBPs-labeled cancer cell while being photothermally killed. The results show distance-related thermal propagation because the temperature increase reaches as high as 10 °C at a distance of 5 μm from the cell, whereas the temperature difference drops rapidly to 5 °C when this distance increases to 15 μm. This approach shows that the photothermal conversion from AuNBPs is sufficient to kill the cancer cells, and the temperature increase can be controlled within the micrometer level at a certain period of time. Overall, we present a micrometer-size thermometer platform and provide an innovative strategy to measure temperature at the micrometer level during photothermal killing of cancer cells.

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.

Single-entity electrochemistry (SEEC), a promising method for biosensing, has an intrinsic limitation on sensitivity since at most one colliding entity can be successfully triggered by one target. Here, we take advantage of one-to-many (1:n) signal amplification to develop a new single-entity electrochemistry biosensing (SEECBS), integrating satellite magnetic nanoparticle (MN)-DNA-Pt nanoparticle (NP) conjugates, duplex-specific nuclease (DSN) assisted Pt NPs releasing with stabilization, and effective collision of small sized and nearly naked Pt NPs. Compared with conventional SEECBS, the 1:n SEECBS can successfully enrich ∼2 nM Pt NPs by adding 50 aM microRNA (miRNA), in other words, ∼4 × 107 Pt NPs can be triggered by one target. The proposed SEECBS allows the detection of 47 aM miRNA-21, nearly 6 orders of magnitude lower than the previous work, and discrimination of nontarget miRNAs containing even single-nucleotide mismatch. Besides, this method has also been successfully demonstrated for quantification of miRNA in different cell lines. Therefore, the proposed method holds great potential for the application of SEECBS in early diagnosis and prognosis monitoring of cancer.

In vivo detection of circulating tumor cells (CTCs) which inspect all of the circulating blood in body seems to have more advantages on cell capture, especially in earlier cancer diagnosis. Herein, based on in vivo microfluidic chip detection system (IV-chip-system), an extracorporeal circulation was constructed to effectively detect and monitor CTCs in vivo. Combined with microfluidic chip and immunomagnetic nanosphere (IMN), this system not only acts as a window for CTC monitoring but also serves as a collector for further cancer diagnosis and research on CTCs. Compared with the current in vivo detection method, this system can capture and detect CTCs in the bloodstream without any pretreatments, and it also has a higher CTC capture efficiency. It is worth mentioning that this system is stable and biocompatible without any irreversible damage to living animals. Taking use of this system, the mimicked CTC cleanup process in the blood vessel is monitored, which may open new insights in cancer research and early cancer diagnosis.

Nanosized oncolytic viral light particles (L-particles), separated from progeny virions, are composed of envelopes and several tegument proteins of viruses, free of nucleocapsids. The noninfectious L-particles experience the same internalization process as mature oncolytic virions, which exhibits great potential to act as targeted therapeutic platforms. However, the clinical applications of L-particle-based theranostic platforms are rare due to the lack of effective methods to transform L-particles into nanovectors. Herein, a convenient and mild strategy has been developed to transform L-particles into near-infrared (NIR) fluorescence Ag2Se quantum dot (QD)-labeled active tumor-targeting nanovectors for real-time in situ imaging and drug delivery. Utilizing the electroporation technique, L-particles can be labeled with ultrasmall water-dispersible NIR fluorescence Ag2Se QDs with a labeling efficiency of ca. 85% and loaded with antitumor drug with a loading efficiency of ca. 87%. Meanwhile, by harnessing the infection mechanism of viruses, viral L-particles are able to recognize and enter tumor cells without further modification. In sum, a trackable and actively tumor-targeted theranostics nanovector can be obtained efficiently and simultaneously. Such multifunctional nanovectors transformed from viral L-particles have exhibited excellent properties of active tumor-targeting, in vivo tumor imaging, and antitumor efficacy, which opens a new window for the development of natural therapeutic nanoplatforms.

The functional groups and π-electron system of carbon dots (C-dots) were carefully controlled by several innovative chemical methods, without any changes in size, to unravel the relationship between the surface structure and photoluminescence (PL). The results of experiments and theoretical calculations reveal that the PL of C-dots is related to the surface state. The energy gap is determined by the coupling of the π-electron system and carbonyl group, and the quantum yield (QY) is dependent on the carbonyl group. The carbonyl group is the main factor increasing the ratio of nonradiation to radiation recombination, thereby leading to the low QY of C-dots. This work provides a strategy for effectively tuning the structure of C-dots, giving rise to the tunable PL emission wavelength and highly desirable QY, which enables us to further unravel the PL mechanism.

Herein, a conceptual approach for significantly enhancing a bead-supported assay is proposed. For the fluorescence imaging technology, optical tweezers are introduced to overcome the fluid viscosity interference and immobilize a single tested bead at the laser focus to guarantee a fairly precise imaging condition. For the selection of fluorescent materials and the signal acquisition means, a type of innovative luminescence confined upconversion nanoparticle with a unique sandwich structure is specially designed to act as an efficient energy donor to trigger the luminescent resonance energy transfer (LRET) process. By further combining the double breakthrough with a molecular beacon model, the newly developed detection strategy allows for achieving a pretty high LRET ratio (≈ 88%) to FAM molecules and offering sound assay performance toward miRNA analysis with a detection limit as low as the sub-fM level, and is capable of well identifying single-base mismatching. Besides, this approach not only is able to accurately qualify the low-abundance targets from as few as 30 cancer cells but also can be employed as a valid cancer early warning tool for performing liquid biopsy.

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.

By using multifunctional optical tweezers (OT) equipped with a 980 nm continuous-wave single-mode diode laser and multiple detectors, we are able to trap single upconversion nanoparticles stably as...

The fluorescence method has made great progress in the construction of sensitive sensors but the background fluorescence of the matrix and photobleaching limit its broad application in clinical diagnosis. Here, we propose a digital single virus immunoassay for multiplex virus detection by using fluorescent magnetic multifunctional nanospheres as both capture carriers and signal labels. The superparamagnetism and strong magnetic response ability of nanospheres can realize efficient capture and separation of targets without sample pretreatment. Due to their distinguishable fluorescence imaging and photostability, the nanospheres enable single-particle counting for ultrasensitive multiplexed detection. Furthermore, the integration of digital analysis provided a reliable quantitative strategy for the detection of rare targets. Based on multifunctional nanospheres and digital analysis, a digital single virus immunoassay was proposed for simultaneous detection of H9N2, H1N1, and H7N9 avian influenza virus without complex signal amplification, whose detection limits were 0.02 pg/mL. Owing to its good specificity and anti-interference ability, the method showed great potential in single biomolecules, multiplexed detection, and early diagnosis of diseases.

Immunoassay is commonly used for the detection of disease biomarkers, but advanced instruments and professional operating are often needed with current techniques. The facile readout strategy for immunoassay is mainly limited to the gold nanoparticles-based colorimetric detection. Here, we show that photothermal nanoparticles can be applied for biosensing and immunoassay with temperature as readout. We develop a plasmonic and photothermal immunoassay that allows straightforward readout by color and temperature based on crystal growth, without advanced equipment. It is demonstrated that alkaline phosphatase-triggered silver deposition on the surface of gold nanostars causes a large blue shift in the localized surface plasmon resonance of the nanosensor, accompanied by photothermal conversion efficiency changes. This approach also allows dual-readout of immunoassays with high sensitivity and great accuracy for the detection of prostate-specific antigen in complex samples. Our strategy provides a promising way for point-of-care testing and may broaden the applicability of programmable nanomaterials for diagnostics.

Rapid and sensitive foodborne pathogen detection assay, which can be applied in multiple fields, is essential to timely diagnosis. Herein, we proposed a multisignal readout lateral flow immunoassay for Salmonella typhimurium ( S. typhi) detection. The assay employs colorimetric-fluorescent-magnetic nanospheres (CFMNs) as labels, which possess multifunctional target separation and enrichment, multisignal readout, and two formats of quantitation. The assay for S. typhi detection involves magnetic separation and chromatography. First, the S. typhi were separated and enriched from matrix by antibody labeled CFMNs, and then the S. typhi-containing suspension is added onto the sample pad to flow up the test strip. The introduction of magnetic separation enhances anti-interference ability and 10-fold sensitivity, making the assay possible for practical application. The assay has realized naked eye detection of 1.88 × 104 CFU/mL S. typhi, and 3.75 × 103 CFU/mL S. typhi can be detected with a magnetic assay reader, which is 2-4 orders of magnitude lower than other label-based LFIAs, with a quantitation range of 1.88 × 104 to 1.88 × 107 CFU/mL by measuring the fluorescence intensity and magnetic signal. Moreover, the successful detection of S. typhi in complex matrix (tap water, milk, fetal bovine serum, and whole blood) indicated its potential application in real samples.

Profiling the heterogeneous phenotypes of individual circulating tumor cells (CTCs) from patients is a very challenging task, but it paves new ways for cancer management, especially personalized anticancer therapy. Herein, we propose a chip-assisted multifunctional-nanosphere system for efficient and reliable biomarker phenotype analysis of individual heterogeneous CTCs. Red fluorescent magnetic biotargeting multifunctional nanospheres and green fluorescent biotargeting nanospheres targeting to two kinds of CTC biomarkers are used for convenient dual-fluorescence labeling of CTCs along with magnetic tags. By integrating magnetic enrichment with a size-selective single-cell-trapping microfluidic chip (SCT-chip), over 90% of CTCs, even when the concentrations is as low as 10 CTCs per milliliter of blood, can be individually trapped at highly ordered micropillars, spatially separated from the minimal residual blood cells. Such single CTCs offer easy-readout fluorescence signals, facilitating efficient identification and reliable phenotype analysis in accordance with their biomarker expressions. Therefore, the phenotypes of breast tumor cells in terms of the expression level of human epidermal-growth-factor receptor 2, an important target of clinical anticancer drugs, are accurately assessed, and over 82% of them can be classified into corresponding cell subpopulations. Furthermore, this system demonstrates successful detection and subpopulation analysis of heterogeneous CTCs from seven breast cancer patients, which provides a promising new means for single-cell profiling of CTC-biomarker phenotypes and guiding of personalized anticancer therapy.

Enrichment and purification of circulating tumor cells (CTCs) from peripheral blood is very critical for their detection and downstream analyses, in which many technologies have achieved good effects. However, efficient and high-purity enrichment of CTCs is still challenging because of their rarity and heterogeneity. Herein, anti-CD45 antibody-modified immunomagnetic nanospheres (IMNs (CD45)) and anti-EpCAM antibody and anti-EGFR antibody cocktail modified IMNs (IMNs (EpCAM & EGFR)) were employed to address the two challenges. The cocktail of anti-EpCAM antibody and anti-EGFR antibody enables capture of tumor cells more efficiently than a single antibody, offsetting the loss of CTCs with EpCAM-negative expression or undergoing epithelial to mesenchymal transition (EMT) potentially. Notably, using IMNs (CD45) to remove the vast majority of white blood cells (WBCs) and IMNs (EpCAM & EGFR) to capture tumor cells successively, our method exhibits excellent enrichment and purification capacity. The purity of t...

Establishment of a stable analytical methodology with high-quality results is an urgent need for screening cancer biomarkers in early diagnosis of cancer. In this study, we incorporate holographic optical tweezers with upconversion luminescence encoding to design an imageable suspension array and apply it to conduct the detection of two liver cancer related biomarkers, carcinoembryonic antigen and alpha fetal protein. This bead-based assay is actualized by forming a bead array with holographic optical tweezers and synchronously exciting the upconversion luminescence of corresponding trapped complex beads fabricated with a simple one-step sandwich immunological recognition. Owing to the fact that these flowing beads are stably trapped in the focal plane of the objective lens which tightly converges the array of the laser beams by splitting a 980 nm beam using a diffraction optical element, a fairly stable excitation condition is achieved to provide reliable assay results. By further taking advantage of the eminent encoding capability of upconversion nanoparticles and the extremely low background signals of anti-Stokes luminescence, the two targets are well-identified and simultaneously detected with quite sound sensitivity and specificity. Moreover, the potential on-demand clinical application is presented by employing this approach to respond the targets toward complex matrices such as serum and tissue samples, offering a new alternative for cancer diagnosis technology.

Carbon nanodots (C-dots) have been widely used in sensing, such as detection of ions, small molecules and biomolecules, based on their photoluminescence (PL) quenching by metal ions. Though C-dots prepared by different methods exhibited various sensitives to metal ions, it is labor intensiveness and time-consuming for selecting synthetic route to obtain C-dots that meet requirements of practical applications. Hence, for the high selective and sensitive applications of C-dots, it is the effective approach to reveal the structure–property relationships in the quenching process. Herein, we present an insight into the mechanism of the PL quenching of C-dots by Cu2+. According to the results of PL, UV–vis absorption, time-resolved PL, and femtosecond transient absorbance measurements, we confirmed that the quenching occurs by a photoinduced electron transfer (PET) process from the photoexcited C-dots to the empty d orbits of Cu2+ combining with C-dots. Meanwhile, through separate protecting functional groups o...

When infecting host cells, influenza virus must move on microfilaments (MFs) at the cell periphery and then move along microtubules (MTs) through the cytosol to reach the perinuclear region for genome release. But how viruses switch from the actin roadway to the microtubule highway remains obscure. To settle this issue, we systematically dissected the role of related motor proteins in the transport of influenza virus between cytoskeletal filaments in situ and in real-time using quantum dot (QD)-based single-virus tracking (SVT) and multicolor imaging. We found that the switch between MF- and MT-based retrograde motor proteins, myosin VI (myoVI) and dynein, was responsible for the seamless transport of viruses from MFs to MTs during their infection. After virus entry by endocytosis, both the two types of motor proteins are attached to virus-carrying vesicles. MyoVI drives the viruses on MFs with dynein on the virus-carrying vesicle hitchhiking. After role exchanges at actin-microtubule intersections, dynein drives the virus along MTs toward the perinuclear region with myoVI remaining on the vesicle moving together. Such a "driver switchover" mechanism has answered the long-pending question of how viruses switch from MFs to MTs for their infection. It will also facilitate in-depth understanding of endocytosis.

Monodisperse hydrophilic quantum dots (QDs) are promising labeling materials for biomedical applications. However, the controllable preparation of monodisperse hydrophilic QDs with amphiphilic polymers remains a challenge. Herein, the molecular structures of amphiphilic polymers assembled on different-sized QDs are investigated. Both the experimental results and the molecular dynamics (MD) calculation suggest that the grafting ratio of amphiphilic polymers assembled on QDs increases as the size of QDs increases. Thus, the controllable preparation of different-sized monodisperse hydrophilic QDs can be achieved by simply varying the grafting ratio of amphiphilic molecules and applied in the simultaneous labeling of three tumor biomarkers.

As an emerging fascinating fluorescent nanomaterial, carbon nanodots (CDs) have attracted much attention owing of their unique properties such as small size, antiphotobleaching, and biocompatibility. However, its use in biomedical analysis is limited because of its low quantum yield. Herein, we constructed a dual amplification fluorescence sensor by incorporating immunohybridization chain reaction (immuno-HCR) and metal-enhanced fluorescence (MEF) of CDs for the detection of alpha fetal protein (AFP). The immunoplasmonic slide and detection antibodies-conjugated oligonucleotide initiator are served to capture and probe AFP molecules, respectively. Then, CD-tagged hairpin nucleic acids were introduced to trigger the HCR, in which the hairpin nucleic acid and oligonucleotide initiator are complementary. The interaction between CDs and the gold nanoisland film greatly improves the radiative decay rate, increases the quantum yield, and enhances the fluorescence emission of the CDs. Furthermore, the HCR provides secondary amplification of fluorescence intensity. Therefore, the MEF-capable immunohybridization reactions provide obvious advantages and result in exceptional sensitivity. In addition, the sandwich immunoassay method offers high specificity. The results show a wide linearity between the fluorescence intensity and AFP concentration over 5 orders of magnitude (0.0005-5 ng/mL), and the detection limit reaches as low as 94.3 fg/mL. This method offers advantages of high sensitivity and reliability, wide detection range, and versatile plasmonic chips, thus presenting an alternative for the technologies in biomedical analysis and clinical applications.

Aptamers have attracted much attention as the next generation of affinity reagents. Unfortunately, the selection efficiency remains a critical bottleneck for the widespread application of aptamers. Herein, to accelerate aptamers discovery, a multifunctional microfluidic selection platform was developed, on which the selection efficiency was greatly improved and high-affinity and -specificity aptamers were generated within two round selections. The multifunctional screening platform, precisely manipulating magnetic beads on the micrometer scale, improved selection performance based on microfluidic continuous flow and enhanced the selection process control via in situ monitoring and real-time evaluation. This method could suppress ∼50-fold nonspecific binding nucleic acids compared to the conventional methods, further eliminate weakly bound nucleic acids within 9 min, and simultaneously perform the negative selection and positive selection. And the selection effectiveness was in situ and real-time monitoring. Three aptamers showed high affinity and specificity toward mucin 1 (MUC1) with dissociation constants (K

Enrichment and purification of bacteria from complex matrices are crucial for their detection and investigation, in which magnetic separation techniques have recently show great application advantages. However, currently used magnetic particles all have their own limitations: Magnetic microparticles exhibit poor binding capacity with targets, while magnetic nanoparticles suffer slow magnetic response and high loss rate during treatment process. Herein, we used a highly controllable layer-by-layer assembly method to fabricate quick-response magnetic nanospheres (MNs), and with Salmonella typhimurium as a model, we successfully achieve their rapid and efficient enrichment. The MNs combined the advantages of magnetic microparticles and nanoparticles. On the one hand, the MNs had a fast magnetic response, and almost 100% of the MNs could be recovered by 1 min attraction with a simple magnetic scaffold. Hence, using antibody conjugated MNs (immunomagnetic nanospheres, IMNs) to capture bacteria hardly generated loss and did not need complex separation tools or techniques. On the other hand, the IMNs showed much excellent capture capacity. With 20 min interaction, almost all of the target bacteria could be captured, and even only one bacterium existing in the samples was not missed, comparing with the immunomagnetic microparticles which could only capture less than 50% of the bacteria. Besides, the IMNs could achieve the same efficient enrichment in complex matrices, such as milk, fetal bovine serum, and urine, demonstrating their good stability, strong anti-interference ability, and low nonspecific adsorption. In addition, the isolated bacteria could be directly used for culture, polymerase chain reaction (PCR) analyses, and fluorescence immunoassay without a release process, which suggested our IMNs-based enrichment strategy could be conveniently coupled with the downstream identification and analysis techniques. Thus, the MNs provided by this work showed great superiority in bacteria enrichment, which would be a promising tool for bacteria detection and investigation.

The 2014-16 Ebola virus (EBOV) outbreak in West Africa has attracted widespread concern. Rapid and sensitive detection methods are urgently needed for diagnosis and treatment of the disease. Here, we propose a novel method for EBOV detection based on efficient amplification of electroluminescent nanospheres (ENs) coupled with immunomagnetic separation. Uniform ENs are made by embedding abundant amounts of CdSe/ZnS quantum dots (QDs) into copolymer nanospheres through simple ultrasound. Compared to QDs, ENs can enhance electroluminescence (ECL) signals by approximately 85-fold, achieving a signal-to-background ratio high enough for EBOV detection. The introduction of magnetic nanobeads (MBs) can selectively separate targets from complex samples, simplifying the operation process and saving time. The presence of MBs can amplify ECL by approximately 3-fold, improving detection sensitivity. By integration of ENs with MBs, a sensitive electroluminescence biosensor is established for EBOV detection. The linear range is 0.02-30 ng/mL with a detection limit of 5.2 pg/mL. This method provides consistent reproducibility, specificity, and anti-interference ability and is highly promising in clinical diagnosis 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.

Number concentration of nanoparticles is a critical and challenging parameter to be identified. Recently, gravimetric strategy is a fundamental method for absolute quantification, which is widely accepted and used by researchers, yet limited by the inaccuracy in measuring related parameters (e.g, density). Hence, we introduced isopycnic gradient centrifugation to determine the nanopartices' density and improved the current gravimetric method for more accuracy. In this work, polymer nanospheres were used as a model to validate this method. Through isopycnic gradient centrifugation, nanospheres finally reached the zone of equal density as them. By measuring the density of the medium solution in this zone, the nanospheres' density was identified. Then, the density was multiplied by the volume of a single nanosphere characterized by transmission electron microscopy (TEM), and the average weight of a single nanosphere was obtained. Using total weight of the nanospheres divided by the unit weight, their number concentration was quantified. Directly using the real density of the nanoparticles achieved more accurate quantification than the current gravimetric method which used the density of the bulk material counterparts for calculation. Besides, compared with the viscosity/light scattering method and the high-sensitivity flow cytometry (HSFCM) method (another two kinds of typical methods respectively based on light measurements and single particle counting), the improved gravimetric method showed better reproducibility and more convenience. Further, we modified the nanospheres with streptavidin (SA) and antibody, and through biorecognition interaction, we determined the amount of the active affinity sites on each biofunctional nanosphere. Moreover, their bioactivity in different storage conditions was monitored, which showed good stability even in PBS at 4 °C over one year. Our work provided a promising method for more accurately determining the absolute number concentration of nanoparticles and the active affinity sites on their surfaces, which would greatly facilitate their downstream applications.

Sensitive and quantitative detection of protein biomarkers with a point-of-care (POC) assay is significant for early diagnosis, treatment, and prognosis of diseases. In this paper, a quantitative lateral flow assay with high sensitivity for protein biomarkers was established by utilizing fluorescent nanospheres (FNs) as reporters. Each fluorescent nanosphere (FN) contains 332 ± 8 CdSe/ZnS quantum dots (QDs), leading to its superstrong luminescence, 380-fold higher than that of one QD. Then a detection limit of 27.8 pM C-reaction protein (CRP) could be achieved with an immunofluorescent nanosphere (IFN)-based lateral flow test strip. The assay was 257-fold more sensitive than that with a conventional Au-based lateral flow test strip for CRP detection. Besides, the fluorescence intensity of FNs and bioactivity of IFNs were stable during 6 months of storage. Hence, the assay owns good reproducibility (intra-assay variability of 5.3% and interassay variability of 6.6%). Furthermore, other cancer biomarkers (PSA, CEA, AFP) showed negative results by this method, validating the excellent specificity of the method. Then the assay was successfully applied to quantitatively detect CRP in peripheral blood plasma samples from lung cancer and breast cancer patients, and healthy people, facilitating the diagnosis of lung cancer. It holds a good prospect of POC protein biomarker detection.

We present an analytical platform by combining near-infrared optical tweezers with two-photon excitation for fluorescence detection of H5N1 virus gene sequences. A heterogeneous enrichment strategy, which involved polystyrene (PS) microsphere and quantum dots (QDs), was adopted. The final hybrid-conjugate microspheres were prepared by a facile one-step hybridization procedure by using PS microspheres capturing target DNA and QDs tagging, respectively. Quantitative detection was achieved by the optical tweezers setup with a low-cost 1064 nm nanosecond pulse laser for both optical trapping and two-photon excitation for the same hybrid-conjugate microsphere. The detection limits for both neuraminidase (NA) gene sequences and hemagglutinin (HA) gene sequences are 16-19 pM with good selectivity for one-base mismatch, which is approximately 1 order of magnitude lower than the most existing fluorescence-based analysis method. Besides, because of the fact that only signal from the trapped particle is detected upon two-photon excitation, this approach showed extremely low background in fluorescence detection and was successfully applied to directly detect target DNA in human whole serum without any separation steps and the corresponding results are very close to that in buffer solution, indicating the strong anti-interference ability of this method. Therefore, it can be expected to be an emerging alternative for straightforward detecting target species in complex samples with a simple procedure and high-throughput.

Labeling and imaging of live cells with quantum dots (QDs) has attracted great attention in the biomedical field over the past two decades. Maintenance of the fluorescence of QDs in a biological environment is crucial for performing long-term cell tracking to investigate the proliferation and functional evolution of cells. The cell-penetrating peptide transactivator of transcription (TAT) is a well-studied peptide to efficiently enhance the transmembrane delivery. Here, we used TAT peptide-conjugated QDs (TAT-QDs) as a model system to examine the fluorescence stability of QDs in live cells. By confocal microscopy, we found that TAT-QDs were internalized into cells by endocytosis, and transported into the cytoplasm via the mitochondria, Golgi apparatus, and lysosomes. More importantly, the fluorescence of TAT-QDs in live cells was decreased mainly by cell proliferation, and the low pH value in the lysosomes could also lower the fluorescence intensity of intracellular QDs. Quantitative analysis of the amount of QDs in the extracellular region and whole cells indicated that the exocytosis was not the primary cause of fluorescence decay of intracellular QDs. This work facilitates a better understanding of the fluorescence stability of QDs for cell imaging and long-term tracking in live cells. Also, it provides insights into the utility of TAT for transmembrane transportation, and the preparation and modification of QDs for cell imaging and tracking.

With unique and tunable photoluminescence (PL) properties, carbon nanodots (CNDs) as a new class of optical tags have been extensively studied. Because of their merits of controllability and sensitivity to the surface of nanomaterials, electrochemical methods have already been adopted to study the intrinsic electronic structures of CNDs. In this review, we mainly deal with the electrochemical researches of CNDs, including preparation, PL mechanism, and biosensing.

Recently, research on carbon nanodots (C-dots), a new type of luminescent nanoparticles with superior optical properties, biocompatibility, and low cost, has been focused on exploring novel properties and structure-related mechanisms to extend their scope. Herein, electrochemiluminescence, a surface-sensitive tool, is used to probe the unrevealed property of carbon nanodots which is characterized by surface oxygen-containing groups. Together with chemiluminescence, carbon nanodots as the coreactants for the anodic electrochemiluminescence of Ru(bpy)3(2+) are demonstrated for the first time. During the anodic scan, the benzylic alcohol units on the C-dots surface are oxidized "homogeneously" by electrogenerated-Ru(bpy)3(3+) to form reductive radical intermediate, which further reduce Ru(bpy)3(3+) into Ru(bpy)3(2+)* that produces a strong ECL emission. This work has provided an insight into the ECL mechanism of the C-dots-involved system, which will be beneficial for in-depth understanding of some peculiar phenomena of C-dots, such as photocatalytic activity and redox properties. Moreover, because of the features of C-dots, the ECL system of Ru(bpy)3(2+)/C-dots is more promising in the bioanalysis.

Manipulating biochemical reactions in living cells to synthesize nanomaterials is an attractive strategy to realize their synthesis that cannot take place in nature. Yeast cells have been skillfully utilized to produce desired nanoparticles through spatiotemporal coupling of intracellular nonrelated biochemical reaction pathways for formation of fluorescent CdSe quantum dots. Here, we have successfully transformed Staphylococcus aureus cells into cellular beacons (fluorescing cells), all of which are highly fluorescent and photostable with perfect uniformity. Importantly, on the basis of such cells, we efficiently fabricated fluorescent nanobioprobes by a specific interaction between the protein A expressed on the S. aureus surface and the Fc fragment domain of antibodies, avoiding the use of other common methods for cell surface modifications, such as molecular covalent connection or more difficult genetic and metabolic engineering. Coupled with immunomagnetic beads, the resulting fluorescent-biotargeting bifunctional cells, i.e., biotargeting cellular beacons, can be employed as nanobioprobes for detection of viruses, bacteria, and tumor cells. With this method, H9N2 AIV can be detected specifically with a limit of 8.94 ng/mL (based on protein content). Furthermore, diverse probes for detection of different pathogens or for other biomedical applications can be easily obtained by simply changing the antibody conjugated to the cell surface.