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BACKGROUND: Cell-surface proteins, which are closely associated with various physiological and pathological processes, have drawn much attention in drug discovery and disease diagnosis. Thus, wash-free imaging of the target cell-surface protein under its native environment is critical and helpful for early detection and prognostic evaluation of diseases. RESULTS: To minimize the interference from autofluorescence and fit the penetration depth towards tissue samples, we developed a fluorogenic antibody-based probe, Ab-Cy5.5, which will liberate > 5-fold turn-on near-infrared (NIR) emission in the presence of its target antigen within 10 min. SIGNIFICANCE: By taking advantage of the fluorescence-quenched dimeric H-aggregation of Cy5.5, Ab-Cy5.5 with Cy5.5 attached at the N-terminus showed negligible background signal, allowing direct imaging of the target cell-surface protein in both living cells and tissue samples without washing.

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Optical bioimaging is an ever-growing field that benefits both from the fast progress of optical instrumentation and modalities, and from the development of light-emitting probes. The efficacy of molecular fluorescent dyes is crucial, yet hindered by limited brightness and hydrophilicity. Addressing these challenges, self-stabilized fluorogenic organic nanoparticles only made of pure dyes (dFONs) are introduced in this work. Comprising thiol-sensitive fluorogenic chromophores, these dFONs exhibit enhanced brightness exclusively in the presence of biological thiols, notably glutathione, overcoming the need for water-solubilizing moieties. Importantly, these nanoparticles demonstrate large fluorescence and one- and two-photon brightness, enabling sensitive bioimaging of intracellular thiols at micromolar concentrations. Notably, only the pristine fluorogenic nanoparticles can penetrate the cells and does not require to wash the cells before imaging, emphasizing their unique role as dye carriers, fluorogenic probes and ease of use. This work highlights the transformative potential of dFONs in advancing optical bioimaging, paving the way for the use of dFONs not just as tracers, but also now as biosensors and ultimately in the future as biomarkers.

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The printing and dyeing industry is currently accelerating toward a direction of high efficiency, energy conservation, environmental protection, and integration with digitalization. Disperse dye wash-free digital inkjet dyeing is a revolutionary breakthrough for cleaning and coloring polyester fabric. Based on the solubility parameters and the hot-melt dyeing characteristics of disperse dyes, soft, hard, and functional monomers of acrylate were used as the main body. Moreover, single-vinyl fluorinated polysiloxane and divinyl polysiloxane with low solubility parameters were used as modified monomers. A modified polyacrylate (PFSMA) adhesive containing silicon in the main chain and fluorine silicon in the side chain was prepared via miniemulsion polymerization. Using disperse digital inkjet dyeing of polyester fabric without washing can realize energy saving, emission reduction, and carbon reduction. Results showed that the optimum preparation conditions of PFSMA were as follows: DVFS molecular weight of 957 g/mol and DVFS content of 2.5 wt %. Compared with that of polyacrylate (PA), the glass-transition temperature of PFSMA film decreased, and its water resistance, toughness, and adhesion enhanced. When the PFSMA content in the wash-free disperse red ink was 8 wt %, the color yields of the front and back of the PFSMA jet-dyed polyester fabric were 18.86 and 13.28, respectively. Moreover, the color yield of the front of PFSMA jet-dyed polyester fabric was 39.9% higher than that of the pure liquid disperse red jet-dyed fabric. The simulated fixation rate was 87.9%, approximately 2.9 times higher than that of the PA wash-free jet-dyed fabric. The color fastness to dry rubbing reached level 4 and the color fastness to wet rubbing reached level 3-4, which was one level higher than that of pure liquid disperse red jet-dyed fabrics. The color fastness to soaping reached grade 5 and the color fastness to heat compression reached grades 4-5 and above. The fabric was a little firmer but smoother. The color properties, color fastness, and hand feeling of the PFSMA wash-free jet-dyed polyester fabric exceeded the levels of commercially available adhesives.

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Both viral infection and vaccination affect the antibody repertoire of a person. Here, we demonstrate that the analysis of serum antibodies generates information not only on the virus type that caused the infection but also on the specific virus variant. We developed a rapid multiplex assay providing a fingerprint of serum antibodies against five different SARS-CoV-2 variants based on a microarray of virus antigens immobilized on the surface of a label-free reflectometric biosensor. We analyzed serum from the plasma of convalescent subjects and vaccinated volunteers and extracted individual antibody profiles of both total immunoglobulin Ig and IgA fractions. We found that Ig level profiles were strongly correlated with the specific variant of infection or vaccination and that vaccinated subjects displayed a larger quantity of total Ig and a lower fraction of IgA relative to the population of convalescent unvaccinated subjects.

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Lipid droplets (LDs) participating in various cellular activities and are increasingly being emphasized. Fluorescence imaging provides powerful tool for dynamic tracking of LDs, however, most current LDs probes remain inconsistent performance such as low Photoluminescence Quantum Yield (PLQY), poor photostability and tedious washing procedures. Herein, a novel yellow-emissive carbon dot (OT-CD) has been synthesized conveniently with high PLQY up to 90%. Besides, OT-CD exhibits remarkable amphiphilicity and solvatochromic property with lipid-water partition coefficient higher than 2, which is much higher than most LDs probes. These characters enable OT-CD high brightness, stable and wash-free LDs probing, and feasible for in vivo imaging. Then, detailed observation of LDs morphological and polarity variation dynamically in different cellular states were recorded, including ferroptosis and other diseases processes. Furthermore, fast whole imaging of zebrafish and identified LD enrichment in injured liver indicate its further feasibility for in vivo application. In contrast to the reported studies to date, this approach provides a versatile conventional synthesis system for high-performance LDs targeting probes, combing the advantages of easy and high-yield production, as well as robust brightness and stability for long-term imaging, facilitating investigations into organelle interactions and LD-associated diseases.

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Mercury ions (Hg2+) primarily target mitochondria in the cells. Therefore, the development of novel probes that specifically target mitochondria in the presence of Hg2+ is of immense importance. Most previously reported probes that utilize the softness of S, Te, O, and/or N atoms for Hg2+ binding often face problems such as fluorescence quenching and off-target signals. In this study, bromide-hydrocarbon pyridinium salts were designed to target the mitochondria and chelate Hg2+ via Hg-Br coordination bonds. As a prototype, four aggregation-induced emission (AIE) fluorogens, namely TPP-Br, TPP-Cl, R1, and R2, with a similar D-π-A structure but slight differences in their halogen substituents, were designed. Among them, only TPP-Br achieved the highly selective and sensitive detection of Hg2+ by triggering its AIE properties, resulting in remarkable emission enhancement (80-fold), colorimetry, and the Tyndall effect. TPP-Br exhibited high selectivity and sensitivity to Hg2+ with a detection limit of 0.35 µM, rapid response time (<10 s), and large Stokes shift of 185 nm. Their interaction modes were studied using a combination of 1H nuclear magnetic resonance spectroscopy, scanning electron microscopy, fluorescent lifetime decay, and theoretical calculations. TPP-Br exhibited a low emission background in cells, whereas in the presence of Hg2+, mitochondria were lit up with wash-free staining. This study provides a powerful tool for accurately diagnosing mercury poisoning-related diseases in mitochondria.

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Prostate-specific membrane antigen (PSMA) imaging probes are a promising tool for the diagnosis and image-guided surgery of prostate cancer (PCa). However, PSMA-specific luminescence probes for PCa detection and heterogeneity studies with high imaging contrast are lacking. Here, we report the first near-infrared (NIR) iridium(III) complex for the wash-free and specific imaging of PSMA in PCa cells and spheroids. The conjugation of a PSMA inhibitor, Lys-urea-Glu, to an iridium(III) complex synergizes the PSMA-specific affinity and biocompatibility of the inhibitor with the desirable photophysical properties of the iridium(III) complex, including NIR emission (670 nm), high photostability and a large Stokes shift. The cellular impermeability of the probe along with its strong binding affinity to PSMA enhances its specificity for PSMA, enabling the washing-free luminescent imaging of membrane PSMA with lower cytotoxicity. The probe was successfully applied for selectively visualizing PSMA-expressing cells and for the imaging of PSMA in a multicellular PCa model with good imaging penetration, indicating its potential use in complicated and heterogeneous tumor microenvironments. Furthermore, the probe showed good imaging performance in the PCa-bearing tumor mice via targeting PSMA in vivo. This work provides a novel strategy for the development of highly sensitive and specific NIR probes for PSMA in biological systems in vitro, which is of great significance for the precise diagnosis of PCa and for elucidating PCa heterogeneity.

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With the aggravated burden of opioid use disorder spreading worldwide, demands for new forms of opioid receptor agonist/antagonist constitute immense research interest. The Mu-opioid receptor (MOR) is currently in the spotlight on account of its general involvement in opioid-induced antinociception, tolerance and dependence. MOR binding assay, however, is often complicated by difficulty in MOR separation and purification, as well as the tedious procedure in standard biolayer interferometry and surface plasmon resonance measurements. To this end, we present TPE2N as a light-up fluorescent probe for MOR, which exhibits satisfactory performance in both live cells and lysates. TPE2N was elaborately designed based on the synergistic effect of twisted intramolecular charge-transfer and aggregation-induced emission by incorporating a tetraphenylethene unit to emit strong fluorescence in a restrained environment upon binding with MOR through the naloxone pharmacore. The developed assay enabled high-throughput screening of a compound library, and successfully identified three ligands as lead compounds for further development.

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Mitochondria play significant roles in maintaining a stable internal environment for cell metabolism. Hence, real-time monitoring of the dynamics of mitochondria is essential for further understanding mitochondria-related diseases. Fluorescent probes provide powerful tools for visualizing dynamic processes. However, most mitochondria-targeted probes are derived from organic molecules with poor photostability, making long-term dynamic monitoring challenging. Herein, we design a novel mitochondria-targeted probe based on carbon dots with high performance for long-term tracking. Considering that the targeting ability of CDs is related to surface functional groups, which are generally determined by the reaction precursors, we successfully constructed mitochondria-targeted O-CDs with emission at 565 nm through solvothermal treatment of m-diethylaminophenol. The O-CDs are bright with a high quantum yield of 12.61%, high mitochondria-targeting ability, and good stability. The O-CDs possess a high quantum yield (12.61%), specific mitochondria-targeting ability, and outstanding optical stability. Owing to the abundant hydroxyl and ammonium cations on the surface, O-CDs showed obvious accumulation in mitochondria with a high colocalization coefficient of up to 0.90 and remained steady even after fixation. Besides, O-CDs showed outstanding compatibility and photostability under various interruptions or long-time irradiation. Therefore, O-CDs are preferable for the long-term tracking of dynamic mitochondrial behavior in live cells. We first observed the mitochondrial fission and fusion behaviors in HeLa cells, and then, the size, morphology, and distribution of mitochondria in physiological or pathological conditions were clearly recorded. More importantly, we observed different dynamics interactions between mitochondria and lipid droplets during the apoptosis and mitophagy processes. This study provides a potential tool for exploring interactions between mitochondria and other organelles, further promoting the research on mitochondria-related diseases.

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To obtain a sensitive, wash-free photoelectrochemical biosensor based on electron mediation between an electrode and a photoredox catalyst (PC) label, unavoidable O2-related reactions should have no effect or be beneficial, and the rate of electron mediation should depend on the distance between the PC label and electrode. A wash-free photoelectrochemical biosensor that (i) combines photoredox catalysis of a PC label with electrochemical reduction of an electron mediator, and (ii) uses a light-blocking multilayer of magnetic microparticles was developed. O2 participates as an electron acceptor in photoredox catalysis; thus, increasing rather than decreasing the electrochemical signal. Upon photoirradiation from the opposite side of a transparent indium tin oxide (ITO) electrode in contact with the solution, the light intensity in the solution is sharply decreased by the light-blocking multilayer, which increases the contribution of affinity-bound PC labels on the ITO electrode to the electrochemical signal compared to that of unbound PC labels in solution. Utilizing eosin Y (EY2-) and Fe(CN)64- as the PC and electron mediator (i.e., electron donor), respectively, enabled rapid redox cycling based on photoredox catalysis combined with electroreduction. The cathodic charge is mainly related to electron transfer from Fe(CN)64- to excited EY2- (Type I photosensitization), rather than energy transfer from excited EY2- to O2, which generates 1O2 (Type II photosensitization). The developed detection scheme was applied to wash-free detection of a model target DNA. Detection limits of ∼200 pM were obtained in both phosphate-buffered saline and serum without washing. The developed scheme enables simple photoelectrochemical detection.

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Fluorescence imaging is a powerful technique for continuous observation of dynamic intracellular processes of living cells. Fluorescent probes bearing a fluorescence switching property associated with a specific recognition or reaction of target biomolecule, that is, stimuli-responsibility, are important for fluorescence imaging. Thus, fluorescent probes continue to be developed to support approaches with different design strategies. When compared with simple intensity-changing fluorescent probes, ratiometric fluorescent probes typically offer the advantage of less sensitivity to errors associated with probe concentration, photobleaching, and environmental effects. For intracellular usage, ratiometric fluorescent probes based on small molecules must be loaded into the cells. Thus, probes having intrinsic fluorescence may obscure a change in intracellular signal if the background fluorescence of the remaining extracellular probes is high. To overcome such disadvantages, it is necessary to minimize the extracellular background fluorescence of fluorescent probes. Here, the design strategy of the latent ratiometric fluorescent probe for wash-free ratiometric imaging using a xanthene dye seminapthorhodafluor (SNARF) as the scaffold of fluorophore is discussed.

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A benzothiazolium-based hemicyanine dye (probe 3) has been synthesized by attaching a morpholine group into a phenyl benzothiazolium skeleton. Probe 3 exhibited interesting photophysical characteristics including red emission (λem ≈600 nm), enhanced Stokes shift (Δλ ≈80 nm) and sensitivity to solvent polarity. Although the probe 3 exhibited almost no emission in aqueous environments (φfl ≈0.002), its fluorescence could be increased by ≈50 fold in organic solvents (φfl ≈0.10), making it possible for live cell imaging under wash-free conditions. Probe 3 exhibited excellent ability to visualize cellular mitochondria and lysosomes simultaneously, as observed from fluorescence confocal microscopy. In addition, probe 3 also exhibited good biocompatibility (calculated LC50 > 20 µM) and high photostability.

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Nowadays, there is an increasing demand for more accessible routine diagnostics for patients with respect to high accuracy, ease of use, and low cost. However, the quantitative and high accuracy bioassays in large hospitals and laboratories usually require trained technicians and equipment that is both bulky and expensive. In addition, the multistep bioassays and long turnaround time could severely affect the disease surveillance and control especially in pandemics such as influenza and COVID-19. In view of this, a portable, quantitative bioassay device will be valuable in regions with scarce medical resources and help relieve burden on local healthcare systems. Herein, we introduce the MagiCoil diagnostic device, an inexpensive, portable, quantitative, and rapid bioassay platform based on the magnetic particle spectrometer (MPS) technique. MPS detects the dynamic magnetic responses of magnetic nanoparticles (MNPs) and uses the harmonics from oscillating MNPs as metrics for sensitive and quantitative bioassays. This device does not require trained technicians to operate and employs a fully automatic, one-step, and wash-free assay with a user friendly smartphone interface. Using a streptavidin-biotin binding system as a model, we show that the detection limit of the current portable device for streptavidin is 64 nM (equal to 5.12 pmole). In addition, this MPS technique is very versatile and allows for the detection of different diseases just by changing the surface modifications on MNPs. Although MPS-based bioassays show high sensitivities as reported in many literatures, at the current stage, this portable device faces insufficient sensitivity and needs further improvements. It is foreseen that this kind of portable device can transform the multistep, laboratory-based bioassays to one-step field testing in nonclinical settings such as schools, homes, offices, etc.

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Pathogenic infections, particularly caused by Gram-positive bacteria (G+), pose a serious threat to human health, and therefore the fast and accurate discrimination of G+ bacteria from Gram-negative bacteria (G-) and fungi is highly desirable. Organic molecules with facile synthesis, robust photostability, good biocompatibility, and high selectivity toward pathogens are urgently needed in the clinical diagnosis and therapy. To this end, herein we report the synthesis of two naphthalimide-based bioprobes named tetraphenylethylene-naphthalimide (TPE-NIM) and triphenylamine-naphthalimide (TPA-NIM) with aggregation-induced emission (AIE) characteristic. First, the staining capacity of the designed AIEgens toward six kinds of bacteria and two kinds of fungi was evaluated. Both TPE-NIM and TPA-NIM showed a high degree of binding/imaging selectivity for G+ bacteria over G- bacteria and fungi via a wash-free protocol. Second, the two AIEgens had the ability to visualize the biofilms formed by G+ bacteria (Staphylococcus aureus) and can quickly track the G+ bacteria (Staphylococcus aureus) in red blood cell suspensions. Third, we have revealed that electrostatic attraction and hydrophobic interaction both contribute to the selective binding of the AIEgens toward G+ bacteria. In view of the high binding/imaging specificity toward G+ bacteria, low hemolysis rates, and low toxicity toward the bacterial cells, these AIEgens can be applied for the clinical detection of pathogenic infections caused by G+ bacteria and broaden the theranostic applications of AIE materials.

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MicroRNAs are widely studied as circulating biomarkers for early stage diagnosis of several diseases. Detection and quantification of miRNAs is currently performed through complex and time consuming procedures. Herein we demonstrate a rapid, multiplex, one-pot detection method based on two-step amplification of the signal measured by Reflective Phantom Interface (RPI) label-free optical biosensor. We achieved sub-pM quantification of different miRNAs in about 1.5 h, through specific capture with surface DNA probes combined to a 35-fold mass amplification by an antibody targeting DNA-RNA hybrids and polyclonal secondary antibody, all performed without washing steps. The assay is the result of a modelling and optimization of the multi-step process that has been made possible by the RPI characterization of each individual interaction involved.

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The design and synthesis of water-soluble phototherapeutic agents with near-infrared (NIR) fluorescence emission is highly desirable for cancer diagnosis and treatment. Here, we report the construction of an amphiphilic perylene-derived photosensitizer, AP. AP shows NIR emission with large Stokes shift (130 nm) and high 1O2 quantum yield (22%). It can self-assemble into nanoparticles in aqueous solution with quenched fluorescence emission due to aggregation-induced quenching. Upon membrane anchoring, AP is able to disassemble into free monomer molecules and specifically "light up" the cell membrane without the usually required washing procedures. Furthermore, AP is subsequently used for the efficient photodynamic therapy against cancer cells and solid tumors. The in vitro and in vivo experiments clearly indicate that AP is suitable for biological imaging and can serve as a promising photosensitizer for tumor suppression.

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Biological signaling pathways are underpinned by protein switches that sense and respond to molecular inputs. Inspired by nature, engineered protein switches have been designed to directly transduce analyte binding into a quantitative signal in a simple, wash-free, homogeneous assay format. As such, they offer great potential to underpin point-of-need diagnostics that are needed across broad sectors to improve access, costs, and speed compared to laboratory assays. Despite this, protein switch assays are not yet in routine diagnostic use, and a number of barriers to uptake must be overcome to realize this potential. Here, we review the opportunities and challenges in engineering protein switches for rapid diagnostic tests. We evaluate how their design, comprising a recognition element, reporter, and switching mechanism, relates to performance and identify areas for improvement to guide further optimization. Recent modular switches that enable new analytes to be targeted without redesign are crucial to ensure robust and efficient development processes. The importance of translational steps toward practical implementation, including integration into a user-friendly device and thorough assay validation, is also discussed.

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Extracellular vesicles (EVs) are emerging as promising biomarkers for cancer diagnosis and therapy. Recognizing low-abundance EVs from clinical samples in an easy-to-operate way is highly desired but remains a challenge. Herein, we established an allosteric probe-initiated dual cycle amplification-assisted CRISPR-Cas12a (AID-Cas) platform for sensitive detection of EVs in a wash-free way. In AID-Cas, the allosteric probe can specifically recognize and bind with target EVs and thus initiate the following dual-cycle amplification. Subsequently, the amplified products were transcribed to generate numerous single-stranded RNAs, which could work as crRNA to trigger the trans-cleavage of CRISPR-Cas12a. Consequently, the proposed approach achieved a good linear response to extracted EVs in a concentration range from 102 to 106 particles/µL. Because of its high sensitivity, together with its wash-free convenience, the proposed strategy could have promising clinical potentials for early diagnosis of cancers.

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Magnetic nanoparticles (MNPs) with proper surface functionalization have been extensively applied as labels for magnetic immunoassays, carriers for controlled drug/gene delivery, tracers and contrasts for magnetic imaging, etc. Here, we introduce a new biosensing scheme based on magnetic particle spectroscopy (MPS) and the self-assembly of MNPs to quantitatively detect H1N1 nucleoprotein molecules. MPS monitors the harmonics of oscillating MNPs as a metric for the freedom of rotational process, thus indicating the bound states of MNPs. These harmonics can be readily collected from nanogram quantities of iron oxide nanoparticles within 10 s. The H1N1 nucleoprotein molecule hosts multiple different epitopes that forms binding sites for many IgG polyclonal antibodies. Anchoring IgG polyclonal antibodies onto MNPs triggers the cross-linking between MNPs and H1N1 nucleoprotein molecules, thereby forming MNP self-assemblies. Using MPS and the self-assembly of MNPs, we were able to detect as low as 44 nM (4.4 pmole) H1N1 nucleoprotein. In addition, the morphologies and the hydrodynamic sizes of the MNP self-assemblies are characterized to verify the MPS results. Different MNP self-assembly models such as classical cluster, open ring tetramer, and chain model as well as multimers (from dimer to pentamer) are proposed in this paper. Herein, we claim the feasibility of using MPS and the self-assembly of MNPs as a new biosensing scheme for detecting ultralow concentrations of target biomolecules, which can be employed as rapid, sensitive, and wash-free magnetic immunoassays.

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In this work, a novel bright yellow fluorescent carbon dots (CDs) was synthesized from N-methyl-1,2-phenylenediamine hydrochloride in ethanol solvent through the solvothermal method. The obtained carbon dots had no fluorescence in water, but could specifically light up in the cell which makes the staining process without washing. Interestingly, the imaging process can be performed by simply shaking the culture with the cells for only 1 min, indicating ultrafast and easy to operate fluorescence imaging. Moreover, the carbon dots with abundant amino functional groups had good lysosome targeting properties (the Pearson's correlation coefficient is 0.92) and could achieve the ultrafast lysosome imaging in cell and zebrafish and monitoring cell apoptosis status. This is the first lysosome targeting carbon dots that allows the combination of a short incubation at the second-level with wash-free process providing great potential for continuous observation lysosome in vivo.

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