Trimodal Multiplexed Lateral Flow Test Strips Assisted with a Portable Microfluidic Centrifugation Device.

During the COVID-19 pandemic, the use of lateral flow assays (LFAs) expanded significantly, offering testing beyond traditional health care. Their appeal lies in the ease of use, affordability, and quick results. However, LFAs often have lower sensitivity and specificity compared with ELISA and PCR tests. Efforts to improve LFAs have increased detection times and complexity, limiting their use in large-scale point-of-care settings. To address this, we propose a novel approach using probes that generate multiple signals to enhance the sensitivity and selectivity. This concept also allows multiplexed LFAs to detect multiple analytes concurrently. We developed a trimodal probe that integrates fluorescence, color, and magnetism into a single nanohybrid. The strong plasmonic absorption and high fluorescence of Au nanoparticles and polymer dots enable qualitative and semiquantitative diagnosis, while the magnetic signal facilitates accurate quantitative measurements. As proof-of-concept targets, we selected CYFRA 21-1 and CA15-3, biomarkers for lung and breast cancer, respectively. This trimodal LFA demonstrated a remarkable detection limit of 0.26 ng/mL for CYFRA 21-1 and 2.8 U/mL for CA15-3. To the best of our knowledge, this is the first platform of a trimodal LFA with multiplexing ability. The platform's accuracy and reliability were validated using clinical serum samples, showing excellent consistency with electrochemiluminescence immunoassay results. This universal concept can be applied to other targets, paving the way for the next-generation LFAs.

The Emergence of Tumor-Initiating Cells in an Advanced Hypopharyngeal Tumor Model Exhibits Enhanced Angiogenesis and Nuclear Factor Erythroid 2-Related Factor 2-Associated Antioxidant Effects.

Aims: Hypopharyngeal cancer (HPC) is associated with the worst prognosis of all head and neck cancers and is typically identified in an advanced stage at the time of diagnosis. While oxidative stress might contribute to the onset of HPC in patients using tobacco or alcohol, the extent of this influence and the characteristics of HPC cells in advanced stage remain to be investigated. In this study, we explored whether HPC cells survived from necrotic xenograft tumors at late stage would display increased tumor resistance along with altered tolerance to oxidative stress. Results: The remnant living HPC cells isolated from a late-stage xenograft tumor, named FaDu ex vivo cells, showed stronger chemo- and radioresistance, tumorigenesis, and invasiveness compared with parental FaDu cells. FaDu ex vivo cells also displayed increased angiogenic ability after re-transplantation in mice visualized by in vivo near infrared-II fluorescence imaging modality. Moreover, FaDu ex vivo cells exhibited significant tumor-initiating cell (TIC)-related properties accompanied by a reduction of the level of reactive oxygen species, which was associated with the upregulation of transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). Interestingly, inhibition of Nrf2 by the RNA interference and the chemical inhibitor could reduce the TIC-related properties of FaDu ex vivo cells. Innovation: Oxidative stress potentially initiates HPC, but elevation of Nrf2-associated antioxidant mechanisms would be essential to mitigate this effect for promoting and sustaining the stemness of HPC at the advanced stage. Conclusion: Present data suggest that the antioxidant potency of advanced HPC would be a therapeutic target for the design of adjuvant treatment. Antioxid. Redox Signal. 41, 505-521.

Ultrabright Dibenzofluoran-Based Polymer Dots with NIR-IIa Emission Maxima and Unusual Large Stokes Shifts for 3D Rotational Stereo Imaging.

Emerging organic molecules with emissions in the second near-infrared (NIR-II) region are garnering significant attention. Unfortunately, achieving accountable organic emission intensity over the NIR-IIa (1300 nm) region faces challenges due to the intrinsic energy gap law. Up to the current stage, all reported organic NIR-IIa emitters belong to polymethine-based dyes with small Stokes shifts (<50 nm) and low quantum yield (QY; ≤0.015%). However, such polymethines have proved to cause self-absorption with constrained emission brightness, limiting advanced development in deep-tissue imaging. Here a new NIR-IIa scaffold based on rigid and highly conjugated dibenzofluoran core terminated by amino-containing moieties that reveal emission peaks of 1230-1305 nm is designed. The QY is at least 10 times higher than all synthesized or reported NIR-IIa polymethines with extraordinarily large Stokes shifts of 370-446 nm. DBF-BJ is further prepared as a polymer dot to demonstrate its in vivo 3D stereo imaging of mouse vasculature with a 1400 nm long-pass filter.

A Novel Injection Protocol Using Voluven®-Assisted Indocyanine Green with Improved Near-Infrared Fluorescence Guidance in Breast Cancer Sentinel Lymph Node Mapping-A Translational Study.

BACKGROUND: Near-infrared (NIR) fluorescence-guided surgery with indocyanine green (ICG) has been demonstrated to provide high sensitivity in sentinel lymph node biopsy (SLNB) for breast cancer but has several limitations, such as unstable pharmacokinetics, limited fluorescence brightness, and undesired diffusion to neighboring tissues. This paper investigates the use of Voluven® as the solvent for ICG fluorescence-guided SLNB (ICG-SLNB). METHODS: The photophysical properties of ICG in water and Voluven® were evaluated in laboratory experiments and in a mouse model. Nine patients with early breast cancer underwent subareolar injection of diluted ICG (0.25 mg/ml) for ICG-SLNB. Six of the nine patients received ICG dissolved in Voluven® (ICG:Voluven®), while three were administered ICG dissolved in water (ICG:water); a repetitive injection-observation protocol was followed for all patients. The mapping image quality was evaluated. RESULTS: Laboratory experiments and in vivo mouse study showed improved fluorescence and better targeting using Voluven® as the solvent. ICG-SLNB with a repetitive injection-observation protocol was successfully performed in all nine patients. ICG:Voluven® administration had an overall better signal-to-background ratio (SBR) in sequential sentinel lymph nodes. The rates of transportation within the lymphatics were also improved using ICG:Voluven® compared with ICG:water. CONCLUSIONS: From basic research to animal models to in-human trial, our study proposes a repetitive injection-observation technique with ICG:Voluven®, which is characterized by better transportation and more stable mapping quality for ICG-SLNB in breast cancer patients.

Hybrid polymer dot-magnetic nanoparticle based immunoassay for dual-mode multiplexed detection of two mycotoxins.

We designed polymer dot-magnetic nanoparticle nanohybrids for signal enhancement in a test strip platform. Besides, the multicolor emissions of the Pdots embed multiplexing ability for this test strip. Two mycotoxins, aflatoxin B1 and zearalenone, were tested with the determined limits of detection of 2.15 ng mL-1 and 4.87 ng mL-1, respectively.

Realization of NIR-II 3D whole-body contour and tumor blood vessels imaging in small animals using rotational stereo vision technique.

Significance: Optical imaging in the second near-infrared (NIR-II, 1000 to 1700 nm) region is capable of deep tumor vascular imaging due to low light scattering and low autofluorescence. Non-invasive real-time NIR-II fluorescence imaging is instrumental in monitoring tumor status. Aim: Our aim is to develop an NIR-II fluorescence rotational stereo imaging system for 360-deg three-dimensional (3D) imaging of whole-body blood vessels, tumor vessels, and 3D contour of mice. Approach: Our study combined an NIR-II camera with a 360-deg rotational stereovision technique for tumor vascular imaging and 3D surface contour for mice. Moreover, self-made NIR-II fluorescent polymer dots were applied in high-contrast NIR-II vascular imaging, along with a 3D blood vessel enhancement algorithm for acquiring high-resolution 3D blood vessel images. The system was validated with a custom-made 3D printing phantom and in vivo experiments of 4T1 tumor-bearing mice. Results: The results showed that the NIR-II 3D 360-deg tumor blood vessels and mice contour could be reconstructed with 0.15 mm spatial resolution, 0.3 mm depth resolution, and 5 mm imaging depth in an ex vivo experiment. Conclusions: The pioneering development of an NIR-II 3D 360-deg rotational stereo imaging system was first applied in small animal tumor blood vessel imaging and 3D surface contour imaging, demonstrating its capability of reconstructing tumor blood vessels and mice contour. Therefore, the 3D imaging system can be instrumental in monitoring tumor therapy effects.

Effect of carbon spacer length on the antibacterial properties of zwitterionic poly(sulfobetaine) type copolymeric brushes and their application in wound healing.

Creating infection resistant polymer brushes possessing antiadhesive, bactericidal and cell-compatible features can be regarded as a promising approach to prevent biomaterial-associated infections. In this work, polysulfobetaine type zwitterionic homo- and copolymer brushes with varying spacer lengths (charge separation distance between zwitterions, n = 3, 6 or 12) were allowed to grow onto a tartaric acid based aliphatic polyester substrate using surface initiated atom transfer radical polymerization. All of the brush modified surfaces were thoroughly characterized and assessed for their anti-infective performances in vitro. Strikingly, a suitable copolymer composition, i.e., polyZ6-co-Z12 (50/50 copolymer of polysulfobetaine methacrylates with 6 and 12 spacer lengths), was observed to inhibit bacterial growth completely and its activity was sustained for a long time (>3 months). Surprisingly, its antibacterial effect was found to be bactericidal, as is evident from live-dead staining of residual dead bacterial cells that can be easily released by exposing the surface to salt solution, thereby regenerating the surface. However, all of the other copolymer as well as homopolymer brushes exhibited bacteriostatic behavior. An attempt was made to understand the peculiar behavior of this particular brush composition. Nevertheless, the biocidal and also protein repellent brush did not display any cytotoxicity towards human cells, making it an ideal substrate to be used as an infection resistant biomedical implant. Animal studies further confirmed that this particular copolymeric brush modified scaffold can be a promising anti-infective wound dressing material with rapid wound healing effects as compared to the unmodified scaffold.

Rational Design of Asymmetric Polymethines to Attain NIR(II) Bioimaging at >1100 nm.

Organic molecules having emission in the NIR(II) region are emergent and receiving enormous attention. Unfortunately, attaining accountable organic emission intensity around the NIR(II) region is hampered by the dominant internal conversion operated by the energy gap law, where the emission energy gap and the associated internal reorganization energy λint play key roles. Up to the current stage, the majority of the reported organic NIR(II) emitters belong to those polymethines terminated by two symmetric chromophores. Such a design has proved to have a small λint that greatly suppresses the internal conversion. However, the imposition of symmetric chromophores is stringent, limiting further development of organic NIR(II) dyes in diversity and versatility. Here, we propose a new concept where as far as the emissive state of the any asymmetric polymethines contains more or less equally transition density between two terminated chromophores, λint can be as small as that of the symmetric polymethines. To prove the concept, we synthesize a series of new polymethines terminated by xanthen-9-yl-benzoic acid and 2,4-diphenylthiopyrylium derivatives, yielding AJBF1112 and AEBF1119 that reveal emission peak wavelength at 1112 and 1119 nm, respectively. The quantum yield is higher than all synthesized symmetric polymethines of 2,4-diphenylthiopyrylium derivatives (SC1162, 1182, 1185, and 1230) in this study. λint were calculated to be as small as 6.2 and 7.3 kcal/mol for AJBF1112 and AEBF1119, respectively, proving the concept. AEBF1119 was further prepared as a polymer dot to demonstrate its in vitro specific cellular imaging and in vivo tumor/bone targeting in the NIR(II) region.

TADF-based NIR-II semiconducting polymer dots for in vivo 3D bone imaging.

Intraoperative fluorescence imaging in the second near-infrared (NIR-II) region heralds a new era in image-guided surgery since the success in the first-in-human liver-tumor surgery guided by NIR-II fluorescence. Limited by the conventional small organic NIR dyes such as FDA-approved indocyanine green with suboptimal NIR-II fluorescence and non-targeting ability, the resulting shallow penetration depth and high false positive diagnostic values have been challenging. Described here is the design of NIR-II emissive semiconducting polymer dots (Pdots) incorporated with thermally activated delayed fluorescence (TADF) moieties to exhibit emission maxima of 1064-1100 nm and fluorescence quantum yields of 0.40-1.58% in aqueous solutions. To further understand how the TADF units affect the molecular packing and the resulting optical properties of Pdots, in-depth and thorough density-functional theory calculations were carried out to better understand the underlying mechanisms. We then applied these Pdots for in vivo 3D bone imaging in mice. This work provides a direction for future designs of NIR-II Pdots and holds promising applications for bone-related diseases.

Development of Stereo NIR-II Fluorescence Imaging System for 3D Tumor Vasculature in Small Animals.

Near-infrared-II (NIR-II, 1000-1700 nm) fluorescence imaging boasts high spatial resolution and deep tissue penetration due to low light scattering, reduced photon absorption, and low tissue autofluorescence. NIR-II biological imaging is applied mainly in the noninvasive visualization of blood vessels and tumors in deep tissue. In the study, a stereo NIR-II fluorescence imaging system was developed for acquiring three-dimension (3D) images on tumor vasculature in real-time, on top of the development of fluorescent semiconducting polymer dots (IR-TPE Pdots) with ultra-bright NIR-II fluorescence (1000-1400 nm) and high stability to perform long-term fluorescence imaging. The NIR-II imaging system only consists of one InGaAs camera and a moving stage to simulate left-eye view and right-eye view for the construction of 3D in-depth blood vessel images. The system was validated with blood vessel phantom of tumor-bearing mice and was applied successfully in obtaining 3D blood vessel images with 0.6 mm- and 5 mm-depth resolution and 0.15 mm spatial resolution. The NIR-II stereo vision provides precise 3D information on the tumor microenvironment and blood vessel path.

Self-healable and anti-freezing ion conducting hydrogel-based artificial bioelectronic tongue sensing toward astringent and bitter tastes.

Numerous efforts have been attempted to mimic human tongue since years. However, they still have limitations because of damages, temperature effects, detection ranges etc. Herein, a self-healable hydrogel-based artificial bioelectronic tongue (E-tongue) containing mucin as a secreted protein, sodium chloride as an ion transporting electrolyte, and chitosan/poly(acrylamide-co-acrylic acid) as the main 3D structure holding hydrogel network is synthesized. This E-tongue is introduced to mimic astringent and bitter mouth feel based on cyclic voltammetry (CV) measurements subjected to target substances, which permits astringent tannic acid (TA) and bitter quinine sulfate (QS) to be detected over wide corresponding ranges of 29.3 mM-0.59 µM and 63.8 mM-6.38 µM with remarkable respective sensitivities of 0.2 and 0.12 wt%-1. Besides, the taste selectivity of this E-tongue is performed in the presence of various mixed-taste chemicals to show its high selective behavior toward bitter and astringent chemicals. The electrical self-healability is shown via CV responses to illustrate electrical recovery within a short time span. In addition, cytotoxicity tests using HeLa cells are performed, where a clear viability of ≥95% verified its biocompatibility. The anti-freezing sensing of E-tongue tastes at -5 °C also makes this work to be useful at sub-zero environments. Real time degrees of tastes are detected using beverages and fruits to confirm future potential applications in food taste detections and humanoid robots.

Bimodal Multiplexed Detection of Tumor Markers in Non-Small Cell Lung Cancer with Polymer Dot-Based Immunoassay.

Semiconducting polymer nanoparticles (Pdots) have been demonstrated to be a promising class of probes for use in fluorometric immunochromatographic test strips (ICTS). The advantages of Pdots in ICTSs include ultrahigh brightness, minimal nonspecific adsorption, and multicolor availability, which together contribute to the high sensitivity, good specificity, and multiplexing ability. These unique properties can therefore circumvent several significant challenges of commercial ICTSs, including insufficient specificity/sensitivity and difficulty in quantitative and multiplexed detection. Here, we developed a colorimetric and fluorescent bimodal readout ICTS based on gold-Pdot nanohybrids for the determination of carcinoembryonic antigen (CEA) and cytokeratin 19 fragment (CYFRA 21-1) expressed abnormally in human blood of non-small-cell lung cancer (NSCLS). The vivid color from Au nanomaterials can be used for rapid qualitative screening (colorimetry) in 15 min, while the bright fluorescence of Pdots is ideal for the advanced quantitative measurements of CEA and CYFRA21-1 concentrations in whole blood samples. This bimodal ICTS platform possesses phenomenal detection sensitivity of 0.07 and 0.12 ng/mL for CYFRA21-1 and CEA, respectively. The accuracy and reliability of this ICTS platform were further evaluated with clinical serum samples from NSCLS patients at different stages, showing good consistency with the results from electrochemiluminescence immunoassay.

Molecular Design of Ultrabright Semiconducting Polymer Dots with High NIR-II Fluorescence for 3D Tumor Mapping.

Fluorescence probes emitting in the second near-infrared (NIR-II, 1000-1700 nm) window with the ability for deep-tissue imaging in mammals herald a new era in surgical methodology. However, the brightness of these NIR-II probes is still far from satisfactory due to their low fluorescence quantum yields (QYs), preventing the observation of high-resolution images such as whole-organ vascular networks in real time. Described here is the molecular engineering of a series of semiconducting polymer dots (Pdots) incorporated with aggregation-induced emission moieties to exhibit the QYs as high as 14% in the NIR-II window. Benefiting from the ultrahigh brightness, a 1400 nm long-pass filter is utilized to realize in vivo 3D tumor mapping in mice. To further understand how the geometrical and electron structures of the semiconducting polymers affect their optical properties, the in-depth and thorough density-functional theory calculations are performed to interpret the experimental results. This study lays the groundwork for further molecular design of highly bright NIR-II Pdots.

Recent Developments in Semiconducting Polymer Dots for Analytical Detection and NIR-II Fluorescence Imaging.

In recent years, semiconducting polymer dots (Pdots) have attracted enormous attention in applications from fundamental analytical detection to advanced deep-tissue bioimaging due to their ultrahigh fluorescence brightness with excellent photostability and minimal cytotoxicity. Pdots have therefore been widely adopted for a variety types of molecular sensing for analytical detection. More importantly, the recent development of Pdots for use in the optical window between 1000 and 1700 nm, popularly known as the "second near-infrared window" (NIR-II), has emerged as a class of optical transparent imaging technology in the living body. The advantages of the NIR-II region over the traditional NIR-I (700-900 nm) window in fluorescence imaging originate from the reduced autofluorescence, minimal absorption and scattering of light, and improved penetration depths to yield high spatiotemporal images for biological tissues. Herein, we discuss and summarize the recent developments of Pdots employed for analytical detection and NIR-II fluorescence imaging. Starting with their preparation, the recent developments for targeting various analytes are then highlighted. After that, the importance of and latest progress in NIR-II fluorescence imaging using Pdots are reported. Finally, perspectives and challenges associated with the emergence of Pdots in different fields are given.

Polymethine-Based Semiconducting Polymer Dots with Narrow-Band Emission and Absorption/Emission Maxima at NIR-II for Bioimaging.

Deep-penetration fluorescence imaging in the second near-infrared (NIR-II) window heralds a new era of clinical surgery, in which high-resolution vascular/lymphatic anatomy and detailed cancerous tissues can be visualized in real time. Described here is a series of polymethine-based semiconducting polymers with intrinsic emission maxima in the NIR-IIa (1300-1400 nm) window and absorption maxima ranging from 1082 to 1290 nm. These polymers were prepared as semiconducting polymer dots (Pdots) in aqueous solutions with fluorescence quantum yields of 0.05-0.18 %, and they demonstrate promising applications in noninvasive through-skull brain imaging in live mice with remarkable spatial resolution as well as signal-to-background contrast. This study offers a platform for future design of NIR-IIa or even NIR-IIb emitting Pdots.

Near-Infrared-II Semiconducting Polymer Dots for Deep-tissue Fluorescence Imaging.

Fluorescence imaging, particularly in the NIR-II region (1000-1700 nm), has become an unprecedented tool for deep-tissue in vivo imaging. Among the fluorescent nanoprobes, semiconducting polymer nanoparticles (Pdots) appear to be a promising agent because of their tunable optical and photophysical properties, ultrahigh brightness, minimal autofluorescence, narrow-size distribution, and low cytotoxicity. This review elucidates the recent advances in Pdots for deep-tissue fluorescence imaging and the facing future translation to clinical use.

FRET-Created Traffic Light Immunoassay Based on Polymer Dots for PSA Detection.

There have been enormous efforts for developing the next generations of fluorometric lateral flow immunochromatographic strip (ICTS) owing to the great advances in fluorescent materials in these years. Here we developed one type of fluorometric ICTS based on ultrabright semiconducting polymer dots (Pdots) in which the traffic light-like signals were created by energy transfer depending on the target concentration. This platform was successfully applied for qualitatively rapid screening and quantitatively precise analysis of prostate-specific antigen (PSA) in 10 min from merely one drop of the whole blood sample. This FRET-created traffic light ICTS possesses excellent specificity and an outstanding detection sensitivity of 0.32 ng/mL for PSA. Moreover, we conducted proof-of-concept experiments to demonstrate its potential for multiplexed detection of cancer biomarkers at the same time in an individual test strip by taking advantage of the traffic light signals. To the best of our knowledge, it is the first model of a traffic light-like immunoassay test strip based on Pdots with multiplexing ability. These results would pave an avenue for designing the next generation of point-of-care diagnostics.

Cyanine-Based Polymer Dots with Long-Wavelength Excitation and Near-Infrared Fluorescence beyond 900 nm for In Vivo Biological Imaging.

Bioimaging in the near-infrared window is of great importance to study the dynamic processes in vivo with deep penetration, high spatiotemporal resolution, and minimal tissue absorption, scattering, and autofluorescence. In spite of the huge progress on the synthesis of small organic fluorophores and inorganic nanomaterials with emissions beyond 900 nm, it remains a tough challenge to synthesize semiconducting polymers with fluorescence over this region. Here, we synthesized a series of heptamethine cyanine-based polymers with both absorption and emission in the near-infrared region. We prepared these polymers as semiconducting polymer dots (Pdots) in pure water with great biocompatibility. The fluorescence quantum yield of the Pdots can be as high as 14% with a full width at half-maximum of 53 nm, and their single-particle brightness is more than 20 times higher than commercial quantum dots or ∼300 times brighter than Food and Drug Administration (FDA)-approved indocyanine green (ICG) dyes. We further demonstrated the use of cyanine-based Pdots for specific cellular labeling and long-term tumor targeting in mice. We anticipate that these cyanine-based ultrabright Pdots could open up an avenue for next generations of near-infrared fluorescent agents.

Ultrabright Fluorescent Polymer Dots with Thermochromic Characteristics for Full-Color Security Marking.

Innovative and scalable security technologies are in high demand to deter increasing counterfeiting in modern society. Here, we report the first example of thermochromic-fluorescent ink based on semiconducting polymer dots (Pdots) by taking advantage of the unique optical properties of Pdots. We designed and synthesized two types of thermochromic molecules and then incorporated them with multicolor fluorescent Pdots. The resulting Pdots exhibited colorimetric and fluorescent dual-readout abilities in response to different temperatures which greatly increase the security level for anticounterfeiting applications. These multifunctional Pdots can be easily doped into flexible substrates or prepared as inks. These full-color inks can be further loaded into marker pens for handwriting or cartridges for inkjet printing with excellent signal-to-background contrast. Moreover, complex and delicate full-color images can be printed on security documents or currency for practical use. We anticipate that this first example of thermoresponsive dual-readout methodology based on Pdots will have broad use in advanced security marking technologies.