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Aqueous phase synthesized ternary I–III–VI2 Quantum dots (QDs) are getting more and more attention in biology researches, for their good biocompatibility and easy-to-adjust fluorescence properties. However, the quantum yield (QY) of these aqueous phase synthesized QDs are often pretty low, which seriously hindered their further applications in this field. In general, the ripening of the QDs helps to enhance their QY, closely related to the ripening temperature. But it is still hard to precisely control the fluorescence performance of the QDs products, due to the difficulties in precise temperature control and cumbersome temperature adjusting operations in batch reactors. Here we proposed an integrated droplet microfluidic chip for the automated and successive AgInS2 QDs synthesis and ripening, with both temperatures controlled independently, precisely but easily. Taking advantage of the space-time transformation of the droplet microfluidic chips, the suitable temperature combination for AgInS2 QDs synthesis and ripening was studied, and the high-performance AgInS2 QDs were obtained. In addition, the reason for the decrease of QY of AgInS2 QDs at higher ripening temperature was also explored.

Single-nanoparticle collision electrochemistry (SNCE) has gradually become an attractive analytical method due to its advantages in analytical detection, such as a fast response, low cost, low sample consumption, and in situ real-time detection of analytes. However, the biological analyte's direct detection based on the SNCE blocking mode has the problems of low sensitivity and specificity. In this work, an SNCE biosensor based on SNCE electrocatalytic strategy was used for the detection of H7N9 AIV. Nucleic acid aptamers were introduced to recognize the target virus (H7N9 AIV). After the recognition event, ssDNA

Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that sphingomyelin (SM)-sequestered cholesterol, but not accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol independent, whereas the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein that activates actin nucleation, is recruited to the IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of the CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry. IMPORTANCE IAV infects cells by harnessing cellular endocytic machineries. A better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in the plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin-coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results provide new insights into IAV infection and the pathway/cargo-specific involvement of the cholesterol pool(s).

Fluorescence quenching of carbon nanodots by metal ions has been extensively applied for the determination of oligonucleotides, proteins, small molecules and metal ions. However, the problem of poor selectivity originating from the coordination of surface oxygen-containing groups to many kinds of metal ions has limited the prosperity of carbon nanodots in detection field. Herein, the specific recognition of carbon nanodots to Hg2+ is controlled by rational regulation of the surface structure of carbon nanodots. Passivation of the surface carboxyl and hydroxyl groups plays a decisive role in inhibiting the binding of metal ions with carbon nanodots. Upon the attachment of Hg2+ specific recognition unit, carbon nanodots exhibited a high selectivity to Hg2+. This work facilitates to rationally design the surface structure of carbon nanodots to obtain the desirable selective recognition ability.

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.

The rice striped stem borer (SSB), Chilo suppressalis Walker, and the rice yellow stem borer (YSB), Scirpophaga incertulas Walker, are two of the most damaging pests of rice plant, whose relative crop damage has changed in recent years. Here, we carried out experiments and surveys to understand the potential impact of field flooding on populations of these species. YSB had a consistently higher mortality rate than SSB in overwintering populations. We show that SSB survived under submersion better than YSB, crawled more strongly than YSB, and escaped more effectively from a waterlogged environment than YSB. These differences may relate partly to the longer abdominal prolegs and thoracic legs of SSB than YSB. These factors likely explain why YSB has a higher mortality rate than SSB in overwintering populations and why the relative importance of YSB is declining in some areas. In addition, the flooding method provides an effective cultural practice for some crops. Our results uncover the reasons why flooding practice is more effective for YSB than SSB. The results also point to an effective control measure for YSB.

Objectives Mezlocillin is used in the treatment of neonatal infectious diseases. However, due to the absence of population pharmacokinetic studies in neonates and young infants, dosing regimens differ considerably in clinical practice. Hence, this study aimed to describe the pharmacokinetic characteristics of mezlocillin in neonates and young infants, and propose the optimal dosing regimen based on the population pharmacokinetic model of mezlocillin. Methods A prospective, open-label pharmacokinetic study of mezlocillin was carried out in newborns. Blood samples were collected using an opportunistic sampling method. HPLC was used to measure the plasma drug concentrations. A population pharmacokinetic model was developed using NONMEM software. Results Ninety-five blood samples from 48 neonates and young infants were included. The ranges of postmenstrual age and birth weight were 29–40 weeks and 1200–4000 g, respectively, including term and preterm infants. A two-compartment model with first-order elimination was developed to describe the population pharmacokinetics of mezlocillin. Postmenstrual age, current weight and serum creatinine concentration were the most important covariates. Monte Carlo simulation results indicated that the current dose of 50 mg/kg q12h resulted in 89.2% of patients achieving the therapeutic target, when the MIC of 4 mg/L was used as the breakpoint. When increasing the dosing frequency to q8h, a dose of 20 mg/kg resulted in 74.3% of patients achieving the therapeutic target. Conclusions A population pharmacokinetic model of mezlocillin in neonates and young infants was established. Optimal dosing regimens based on this model were provided for use in neonatal infections.

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 (Ca

Recurrent renal cell carcinoma(reRCC) is associated with poor prognosis and the underlying mechanism is not yet clear. A comprehensive understanding of tumor microenvironment (TME) of reRCC may aid in designing effective anticancer therapies, including immunotherapies. Single-cell transcriptomics holds great promise for investigating the TME, however, this technique has not been used in reRCC. Here, we aimed to explore the difference in the TME and gene expression pattern between primary RCC (pRCC) and reRCC at single-cell level.We performed single-cell RNA sequencing analyses of 32,073 cells from 2 pRCC, 2 reRCC, and 3 adjacent normal kidney samples. 41 pairs of pRCC and reRCC samples were collected as a validation cohort to assess differences observed in single-cell sequencing. The prognostic significance of related cells and markers were studied in 47 RCC patients underwent immunotherapy. The function of related cells and markers were validated via in vitro and in vivo experiments.reRCC had reduced CD8We delineated the heterogeneity of reRCC and highlighted an innovative mechanism that CAFs acted as a suppressor of CD8

Accurate size analysis of nanoparticles (NPs) is vital for nanotechnology. However, this cannot be realized based on conventional single-nanoparticle collision (SNC) because the current intensity, a thermodynamic parameter of SNC for sizing NPs, is always smaller than the theoretical value due to the effect of NP movements on the electrode surface. Herein, a size-dependent dynamic parameter of SNC, current lifetime, which refers to the time that the current intensity decays to 1/e of the original value, was originally utilized to distinguish differently sized NPs. Results showed that the current lifetime increased with NP size. After taking the current lifetime into account rather than the current intensity, the overlap rates for the peak-type current transients of differently sized Pt NPs (10 and 15 nm) and Au NPs (18 and 35 nm) reduced from 73 and 7% to 45 and 0%, respectively, which were closer to the theoretical values (29 and 0%). Hence, the proposed SNC dynamics-based method holds great potential for developing reliable electrochemical approaches to evaluate NP sizes accurately.

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) ...

Cell-derived microvesicles (MVs) are secreted from almost all kinds of mammalian cells into the extracellular space, and play crucial roles in intercellular communication and transporting biomolecules between cells. However, there is a great challenge for visualizing and monitoring of MVs’ bio-behaviors due to the limitations of existing labeling methods. Herein, we report the first paradigm of designer cell-self-implemented labeling of MVs secreted from living mammalian MCF-7 cells in situ using the intracellular-synthesized fluorescent quantum dots (QDs) during the formation of MVs. By elaborately coupling intracellular biochemical reactions and metabolism pathways, the MCF-7 cells can be illuminated brightly by intracellular-biosynthesized fluorescent CdSe QDs. Simultaneously, intracellular-synthesized QDs can be in situ encapsulated by the secreted MVs budding from the plasma membrane of the fluorescing cells to label the MVs with an efficiency of up to 89.9%. The whole labeling process skillfully combines designer precise cell-tuned intricate synthesis of CdSe QDs with mild in-situ labeling via cell-self-implementation just after feeding the cell with suitable chemicals, which is structure- or function-nondestructive and much more straightforward and milder than those by chemical conjugation or indirect encapsulation with conventional fluorogenic labels.

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.

The rapid transmission of vaccinia virus (VACV) in vivo is thought to be closely related to the cell migration induced by it. Cell migration involved in dynamic changes of cell-substrate adhesion and actin cytoskeleton organization, which can influence by the micro/nano-scale topographic structures that cells are naturally exposed to via contact guidance. However, migration behaviors of VACV-infected cells exposed to topographic cues are still unknown. Herein, we designed an open chip with microgrooved poly(dimethyl siloxane) (PDMS) substrate to explore the topography roles in VACV-induced cell migration. Differed from the random cell migration observed in traditional scratch assay on planar substrate, VACV-infected cells had a tendency to persistently migrate along the axis parallel to microgroove with increased velocity. Moreover, infected cells exhibited a dominant elongated protrusion aligned to the micro-grating axis compare to the shorter lamella extended in any direction on smooth substrate. Interestingly, the Golgi complex preferred to relocate behind the nucleus confined within the micro-grating axis in majority of infected migratory cells. The directional polarization of cells embodied in protrusion formation and Golgi reorientation was responsible for the directionally persistent migration behaviors induced by VACV on microgrooved substrate. Infected cells response to substrate topography, causing the actin-filled stretched protrusion containing numerous virions and accelerated movement is likely to facilitate direct and rapid spread of VACV. This work opens a window for us to understand the migration behaviors of infected cells in vivo, and also provides a cue for revealing the relationship between virus-induced cell migration and virus rapid spread.

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.

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.

Among the numerous forms of cancer immunotherapy, cancer vaccines have attracted increasing attention because of their ability to elicit sustained antitumor immune responses and durable tumor regression. Here, a personalized gel-droplet monocyte vaccine (GEMA) derived from host blood was reported. A streamlined microfluidic vaccine production platform was designed to combine the separation of monocytes from host blood and the encapsulation of monocytes in an alginate gel droplet, which simplified the handling of the blood product and permitted the rapid preparation of vaccines. In addition, the application of alginate gel encapsulation not only improved the efficiency of antigen uptake by monocytes, but it also promoted the production of antigen-specific CD8+ T cells in the spleen, resulting in an intense cytotoxic T lymphocyte (CTL) response. Moreover, depending on the disease profile of a specific patient, different adjuvant- and antigen-loaded monocytes could be simultaneously encapsulated in gel droplets to prepare a cocktail vaccine based on patient needs. In this study, anti-PD-1 antibodies were encapsulated in gel droplets as a model adjuvant to obtain a cocktail vaccine, and this demonstrated enhanced antitumor efficacy in a 4T1 breast tumor model. In summary, this study provided a unique vaccine production strategy and an efficient combination therapy approach, holding great promise for the development of personalized cancer vaccines.

The surface of nanocrystals plays a dominant role in many of their physical and chemical properties. However, controllability and tunability of nanocrystal surfaces remain unsolved. Herein, we report that the surface chemistry of nanocrystals, such as near-infrared Ag

Limited to the accuracy of size resolution, single entity collision biosensing (SECBS) for multiplex immunoassays remains challenging, because it is difficult to get the true value of nanoparticle (NP) sizes based on the current intensity due to the complex movement of NPs on the electrode surface. Considering that the size-dependent movement of NPs meanwhile will generate a characteristic current shape, in this work, the huge difference in the current rise time of 5 and 15 nm Pt NPs colliding on an Au ultramicroelectrode (d = 30 μm) was originally used to develop a size-resolved SECBS for multiplex immunoassays of miRNAs. The limit concentration that can be detected was 0.5 fM. Compared with conventional electrochemical biosensors for multiplex immunoassays, for the size-resolved SECBS, one does not need to worry about potential overlapping. Therefore, the proposed method demonstrates a promising potential for the application of SECBS in multiplex immunoassays.

A donor-cell-assisted membrane biotinylation strategy was used to modify small extracellular vesicles (sEVs) while minimizing protein damage, and allowed the sEVs to be loaded onto carriers. Biotinylated programmed death-ligand 1 (PD-L1) positive sEVs were used to select for aptamers from a DNA library. PD-L1 negative sEVs from a homologous cell line were found to remove non-specific aptamer sequences to increase the specificity. After just four rounds, high-affinity aptamers for PD-L1 positive sEVs were selected as novel affinity reagents.

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.

Virus spread is closely related to pathogenesis. Traditional research methods of virus spread do not distinguish cell-to-cell spread from cell-free spread. The uncertainty of virus induced plaque and virus spread direction makes it difficult to track the spread of virus in situ. Herein, the cellular network was fabricated on the basis of PEG modification and soft lithography. Therein, cell numbers and spatial distributions were precisely controlled, e.g., cellular networks like “WHU” can be achieved. Furthermore, a micro-injector was combined with the cellular network for virus spread on the fixed point, in which virus spread direction was limited in one dimension. The results suggested that controllable and flexible cellular network can be constructed on the PEG pattern. The synergia of micro-injector and cellular network provides an advanced tool to investigate virus cell-to-cell spread at the initial infection stage.

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.

Summary Background Identification of high-risk localised renal cell carcinoma is key for the selection of patients for adjuvant treatment who are at truly higher risk of reccurrence. We developed a classifier based on single-nucleotide polymorphisms (SNPs) to improve the predictive accuracy for renal cell carcinoma recurrence and investigated whether intratumour heterogeneity affected the precision of the classifier. Methods In this retrospective analysis and multicentre validation study, we used paraffin-embedded specimens from the training set of 227 patients from Sun Yat-sen University (Guangzhou, Guangdong, China) with localised clear cell renal cell carcinoma to examine 44 potential recurrence-associated SNPs, which were identified by exploratory bioinformatics analyses of a genome-wide association study from The Cancer Genome Atlas (TCGA) Kidney Renal Clear Cell Carcinoma (KIRC) dataset (n=114, 906 600 SNPs). We developed a six-SNP-based classifier by use of LASSO Cox regression, based on the association between SNP status and patients' recurrence-free survival. Intratumour heterogeneity was investigated from two other regions within the same tumours in the training set. The six-SNP-based classifier was validated in the internal testing set (n=226), the independent validation set (Chinese multicentre study; 428 patients treated between Jan 1, 2004 and Dec 31, 2012, at three hospitals in China), and TCGA set (441 retrospectively identified patients who underwent resection between 1998 and 2010 for localised clear cell renal cell carcinoma in the USA). The main outcome was recurrence-free survival; the secondary outcome was overall survival. Findings Although intratumour heterogeneity was found in 48 (23%) of 206 cases in the internal testing set with complete SNP information, the predictive accuracy of the six-SNP-based classifier was similar in the three different regions of the training set (areas under the curve [AUC] at 5 years: 0·749 [95% CI 0·660–0·826] in region 1, 0·734 [0·651–0·814] in region 2, and 0·736 [0·649–0·824] in region 3). The six-SNP-based classifier precisely predicted recurrence-free survival of patients in three validation sets (hazard ratio [HR] 5·32 [95% CI 2·81–10·07] in the internal testing set, 5·39 [3·38–8·59] in the independent validation set, and 4·62 [2·48–8·61] in the TCGA set; all p Interpretation Our six-SNP-based classifier could be a practical and reliable predictor that can complement the existing staging system for prediction of localised renal cell carcinoma recurrence after surgery, which might enable physicians to make more informed treatment decisions about adjuvant therapy. Intratumour heterogeneity does not seem to hamper the accuracy of the six-SNP-based classifier as a reliable predictor of recurrence. The classifier has the potential to guide treatment decisions for patients at differing risks of recurrence. Funding National Key Research and Development Program of China, National Natural Science Foundation of China, Guangdong Provincial Science and Technology Foundation of China, and Guangzhou Science and Technology Foundation of China.

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.

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.