Background Autofluorescence Research Articles

Cyclometalated Iridium(III) Schiff Base Complexes for Chemiluminogenic Bioprobes

Chemiluminogenic bioimaging has emerged as a promising paradigm due to its independence from light excitation, thereby circumventing challenges related to light penetration depth and background autofluorescence. However, the availability of effective chemiluminophores remains limited, which substantially impedes their bio‐applications. Herein, we discovered for the first time that cyclometalated iridium(III) Schiff base complexes can unexpectedly generate chemiluminescence. Notably, the chemiluminescence reaction was rapid, with a half‐life of only 0.86 s, significantly faster than previously reported examples. Unlike conventional chemiluminescent scaffolds, the distinguishing feature of the chemiluminogenic iridium(III) complex is its unique intramolecular imine‐to‐amide conversion upon reaction with reactive oxygen species (ROS). Intriguingly, the chemiluminogenicity of these complexes is not influenced by the cyclometalating ligands but is closely associated with the Schiff base ligand, allowing for tuning of the emission colors via altering the cyclometalating ligands. Additionally, we formulated one of the Schiff base complexes (1) as water‐soluble chemiluminogenic nanoparticles (CLNPs) and successfully employed them as activatable chemiluminescence bioprobes for precise and rapid imaging of hypochlorite‐related biological events both in vitro and in vivo. We believe that this significant finding of the development of chemiluminogenic Schiff base complexes will greatly facilitate the designing of innovative chemiluminophores for theranostic applications.

Cyclometalated Iridium(III) Schiff Base Complexes for Chemiluminogenic Bioprobes.

Chemiluminogenic bioimaginghasemerged as a promisingparadigm due to its independence from light excitation,thereby circumventing challengesrelated to light penetration depth and background autofluorescence. However,the availability of effective chemiluminophoresremains limited,which substantially impedestheir bio-applications. Herein, we discovered for the first time that cyclometalated iridium(III) Schiff base complexes canunexpectedly generate chemiluminescence. Notably, the chemiluminescence reaction was rapid, with a half-life of only0.86s,significantly faster thanpreviously reported examples. Unlike conventional chemiluminescent scaffolds, the distinguishing feature of the chemiluminogeniciridium(III)complex is itsunique intramolecular imine-to-amide conversion upon reaction with reactive oxygen species (ROS). Intriguingly,the chemiluminogenicity of these complexes is not influenced by the cyclometalating ligands but isclosely associated with the Schiff baseligand, allowing for tuning of the emission colors viaaltering the cyclometalating ligands. Additionally,we formulatedone of the Schiff base complexes(1) as water-soluble chemiluminogenic nanoparticles (CLNPs)and successfullyemployedthemas activatable chemiluminescence bioprobes forprecise and rapidimaging of hypochlorite-related biological eventsboth in vitroand in vivo.Webelieve that this significantfinding of the development of chemiluminogenic Schiff basecomplexes will greatlyfacilitate the designingof innovative chemiluminophores for theranostic applications.

Ultrasound-Activated Near-Infrared-II Afterglow Luminescence for Precise Cancer Imaging.

Afterglow fluorescence imaging has been extensively assessed in ultrasensitive bioimaging. Since it eliminates the need for real-time excitation light and thereby circumvents the autofluorescence background of tissue, it holds tremendous potential in accurate biomedical imaging. However, current afterglow probes are rare and emit light only in the visible to near-infrared (NIR) range, which is inadequate for in vivo imaging. To resolve this issue, an ultrasound (US)-activated NIR-II afterglow luminescence probe (NPs-Ce4-SN) emitting afterglow luminescence with a peak at ∼1100 nm was developed. This peak is nearly 400 nm red-shifted compared with other reported afterglow probes. Of note, after US termination, NPs-Ce4-SN undergoes energy transformation to produce 1O2 and subsequently undergoes internal oxidation-reduction reaction to produce NIR-II afterglow, generating high signal-to-noise ratio and high-penetration depth imaging. In vitro and in vivo NIR-II afterglow imaging experiments revealed that NPs-Ce4-SN has good biocompatibility and deep tissue penetration depth, suggesting a diagnostic strategy for in vivo tumor imaging with a high signal-to-noise ratio.

Spectral scanning and fluorescence lifetime imaging microscopy (FLIM) enable separation and characterization of C. elegans autofluorescence in the cuticle and gut.

Caenorhabditis elegans gut and cuticle produce a disruptive amount of autofluorescence during imaging. Although C. elegans autofluorescence has been characterized, it has not been characterized at high resolution using both spectral and fluorescence lifetime-based approaches. We performed high resolution spectral scans of whole, living animals to characterize autofluorescence of adult C. elegans. By scanning animals at 405 nm, 473 nm, 561 nm, and 647 nm excitations, we produced spectral profiles that confirm the brightest autofluorescence has a clear spectral overlap with the emission of green fluorescent protein (GFP). We then used fluorescence lifetime imaging microscopy (FLIM) to further characterize autofluorescence in the cuticle and the gut. Using FLIM, we were able to isolate and quantify dim GFP signal within the sensory cilia of a single pair of neurons that is often obscured by cuticle autofluorescence. In the gut, we found distinct spectral populations of autofluorescence that could be excited by 405 nm and 473 nm lasers. Further, we found lifetime differences between subregions of this autofluorescence when stimulated at 473 nm. Our results suggest that FLIM can be used to differentiate biochemically unique populations of gut autofluorescence without labeling. Further studies involving C. elegans may benefit from combining high resolution spectral and lifetime imaging to isolate fluorescent protein signal that is mixed with background autofluorescence and to perform useful characterization of subcellular structures in a label-free manner.

Nanoparticle-enhanced near-infrared fluorescence probes as a new frontier in cancer imaging

Recent advancements in nanotechnology have significantly enhanced the development of near-infrared fluorescence (NIRF) probes, positioning them as powerful tools in cancer imaging. This paper explores the unique advantages of nanoparticles incorporating NIR dyes, such as indocyanine green (ICG) and DiR, which exhibit deep tissue penetration and minimal background autofluorescence. The enhanced permeability and retention (EPR) effect facilitates selective accumulation in tumor tissues, enabling sophisticated imaging and precision-targeted drug delivery systems. This review highlights the remarkable potential of NIRF imaging techniques in molecular diagnostics, emphasizing their ability to differentiate malignant tissues at a molecular level. Additionally, we discuss various NIRF dye classifications, including cyanine and BODIPY-based probes, along with the development of multifunctional agents that enhance imaging specificity and therapeutic effectiveness. The integration of advanced targeting capabilities, including the use of antibodies and small molecules, further improves the precision of these imaging agents. While challenges remain regarding the pharmacokinetics and potential toxicity of nanoparticle-based probes, their capacity for real-time tumor tracking and the promise of multimodal imaging approaches underscore their transformative role in cancer diagnostics and treatment. By advancing the field of theranostics, nanoparticle-enhanced NIRF probes pave the way for personalized medicine and improved patient outcomes in oncology.

Fused-fiber-photometry based on a low-autofluorescence wideband multimode circulator

Analysis of fluorescent signals in biological samples is widely used in the fields of life science and medicine and can be achieved through conceptually distinct approaches. Here, we report the use of a wideband multimode circulator (WMC), a three-port optical fused-fiber device with an asymmetrical structure specifically designed to ensure efficient light transmission in both illumination and collection paths. It features a flat spectral response and is ideally suited for simultaneous excitation and detection of fluorescence in biological tissue. The WMC used in this work was further improved by minimizing its inherent background autofluorescence. Using the WMC, we demonstrate improved fluorescence detection against already existing fiber-based approaches. Finally, we utilize the WMC to establish fiber photometry recordings in vivo to infer neuronal activity in a small neuromodulatory nucleus in the brainstem of an awake mouse.

Systematic comparison of fluorescence imaging in the near-infrared and shortwave-infrared spectral range using clinical tumor samples containing cetuximab-IRDye800CW.

Shortwave-infrared (SWIR) imaging is reported to yield better contrast in fluorescence-guided surgery than near-infrared (NIR) imaging, due to a reduction in scattering. This benefit of SWIR was shown in animal studies, however not yet in clinical studies with patient samples. We investigate the potential benefit of SWIR to NIR imaging in clinical samples containing cetuximab-IRDye800CW in fluorescence-guided surgery. The potential of the epidermal growth factor-targeted NIR dye cetuximab-IRDye800CW in the shortwave range was examined by recording the absorption and emission spectrum. An ex vivo comparison of NIR and SWIR images using clinical tumor samples of patients with penile squamous cell carcinoma (PSCC) and head and neck squamous cell carcinoma (HNSCC) containing cetuximab-IRDye800CW was performed. The comparison was based on the tumor-to-background ratio and an adapted contrast-to-noise ratio (aCNR) using the standard of care pathology tissue assessment as the golden standard. Based on the emission spectrum, cetuximab-IRDye800CW can be detected in the SWIR range. In clinical PSCC samples, overall SWIR imaging was found to perform similarly to NIR imaging (NIR imaging is better than SWIR in the 2/7 criteria examined, and SWIR is better than NIR in the 3/7 criteria). However, when inspecting HNSCC data, NIR is better than SWIR in nearly all (5/7) examined criteria. This difference seems to originate from background autofluorescence overwhelming the off-peak SWIR fluorescence signal in HNSCC tissue. SWIR imaging using the targeted tracer cetuximab-IRDye800CW currently does not provide additional benefit over NIR imaging in ex vivo clinical samples. Background fluorescence in the SWIR region, resulting in a higher background signal, limits SWIR imaging in HNSCC samples. However, SWIR shows potential in increasing the contrast of tumor borders in PSCC samples, as shown by a higher aCNR over a line.

Advances in Lanthanide-Based NIR-IIb Probes for In Vivo Biomedical Imaging.

The past decades have witnessed the significant development and practical interest of in vivo biomedical imaging technologies and optical materials in the second-near infrared (NIR-II, 1000-1700 nm) window. Imaging with the extended emission wavelength toward the long-wavelength end (NIR-IIb, 1500-1700 nm) further offers micrometer imaging resolution and centimeter tissue penetration depth by taking advantage of the much-reduced photon scattering and near-zero tissue autofluorescence background, which have become a very hot research area. This review focuses on the recent advances in the development of lanthanide-based NIR-IIb probes for in vivo biomedical applications. The progress including ratiometric imaging, multiplexed imaging for wide-field and microscopy, lifetime multiplexing and sensing, persistent luminescence, and multimodal imaging is summarized. Challenges and future directions concerning the investigation of the photophysical and photochemical properties of NIR-IIb probes, the selection of near-infrared cameras as well as the potential extension of the NIR-IIb imaging sub-window are pointed out. This review will inspire readers who have a strong interest in developing optical imaging technology and long-wavelength fluorescence probes for high-contrast in vivo biomedical applications.

Novel Endoplasmic Reticulum-Targeted Luminescent Probe for Visualization of Carbon Monoxide in Drug-Induced Liver Injury.

Drug-induced liver injury (DILI) is a major hepatic dysfunction commonly caused by hepatotoxic drug overdose, resulting in a considerable number of fatalities worldwide. Recent studies have highlighted the regulatory and hepatoprotective effects of carbon monoxide (CO) during the liver injury process. However, precisely tracking the dynamic changes in the composition of CO in DILI is still a great challenge. In this work, leveraging the innovative "quencher-insertion" strategy, a unique endoplasmic reticulum (ER)-targetable lanthanide complex-based luminescence probe, ER-ANBTTA-Eu3+/Tb3+, has been developed for the selective and accurate monitoring of CO fluxes in live cells and laboratory animals. The new probe is composed of three covalently linked functional moieties: the terpyridine polyacid-Eu3+/Tb3+-mixed chelates as the long-lived luminophore, a p-toluenesulfonamide moiety as the ER-anchoring motif, and an allyloxy-nitrobenzyl ether moiety as the CO-specific recognition unit. Upon reaction with CO in the presence of Pd2+ ions, the Tsuji-Trost reaction leads to the cleavage of the allyloxy-nitrobenzyl group from the Eu3+/Tb3+-mixed chelates, which results in the restoration of Tb3+ emission at 538 nm and the attenuation of Eu3+ emission at 688 nm, leading to a dramatic increase of the I538/I688 ratio. In addition to the exceptional response sensitivity and selectivity toward CO, ER-ANBTTA-Eu3+/Tb3+ also exhibits the outstanding ER-locating capability, which allows the probe to be used for imaging of CO in the ER of live cells. Using this probe, combined with the time-gated luminescence imaging mode, the exogenous and endogenous CO in ER of live cells were monitored without the interference of background autofluorescence. Moreover, the upregulation of hepatic CO in DILI mice was successfully visualized. The results suggested the potential of ER-ANBTTA-Eu3+/Tb3+ for deeply exploring the functions of CO in DILI pathogenesis.

Impact of fixation duration on messenger RNA detectability in human formalin-fixed paraffin-embedded brain tissue.

Technologies to study mRNA in post-mortem human brain samples have greatly advanced our understanding of brain pathologies. With ongoing improvements, particularly in formalin-fixed paraffin-embedded tissue, these technologies will continue to enhance our knowledge in the future. Despite various considerations for tissue and mRNA quality, such as pre-mortem health status and RNA integrity, the impact of the tissue fixation time has not been addressed in a systemic fashion yet. In this study, we employed RNAscope to assess mRNA detectability in human post-mortem brain tissue in relation to fixation time. Our results reveal a dynamic change in mRNA detection across varying fixation durations, accompanied by an increase in signal derived from the negative probe and autofluorescence background. These findings highlight the critical relevance of standardized fixation protocols for the collection of human brain tissue in order to probe mRNA abundancy to ensure reliable and comparable results.

Water-Soluble Chitosan-Europium Hybrid Sensor for Singlet Oxygen Detection.

The ability to effectively monitor singlet oxygen (1O2) with fluorescence probes in biological systems is severely restricted mainly by the background autofluorescence of these systems. Though the application of lanthanide complexes as 1O2 monitors successfully resolves this problem with time-gated luminescence measurements, the insolubility of these complexes in an aqueous medium heavily limits their application in biological systems. Here, we present a water-soluble 1O2 sensor based on a chitosan-europium hybrid material. A procedure for the modification of chitosan to expand its solubility to neutral and basic pH, while maintaining its free active amine groups, is described. These are then coupled covalently to a europium-based probe for the detection of 1O2. The resulting hybrid sensor is readily soluble in water across the pH scale and efficiently signals the presence of 1O2 at physiological pH, with the characteristic Eu3+ emission at 611 nm yielding up to a 15-fold increase in emission intensity and a decay time of 332 μs. Being of particular interest for time-gated measurements, this long decay time, coupled with the biocompatibility of chitosan, describes a material with potential biological applications, where 1O2 plays a vital role.

Near-Infrared Unimolecular Chemiluminescence Probes for Deep-Tissue Imaging.

Chemiluminescence (CL) imaging has emerged as a promising optical imaging technique due to minimal background autofluorescence and being excitation-free. However, the emission of most chemiluminescent probes was concentrated in the visible light region, which limited the tissue penetration. Although some NIR chemiluminescence probes have been reported based on the chemiluminescence resonance energy transfer (CRET) strategy, the energy loss was inevitable. Thus, it is crucial to develop near-infrared (NIR) unimolecular probes with direct chemiluminescence. Herein, we propose a strategy of increasing conjugation for designing and synthesizing novel NIR chemiluminescence unimolecular probes that consist of luminol, electron acceptor, π-bridge, and electron donor. Luminol was conjugated to the unimolecular backbone to produce direct NIR chemiluminescence. Notably, the direct CL mechanism of probes was investigated. Compared with CRET-based chemiluminescence, this direct CL was more advantageous to immediately convert the chemical energy into chemiluminescence, avoiding energy degradation. Furthermore, the corresponding nanoparticles with great biosafety were prepared by self-assembly with amphiphilic DSPE-PEG. Especially, TTBL@PEG-NPs with NIR-I emission were successfully used in the sensitive in vivo chemiluminescence imaging of various inflammation models, such as peritonitis, ear swelling, and colitis. This study paves the way for the design of NIR unimolecular chemiluminescence probes and deep-tissue imaging.

Wavefront shaping and imaging through a multimode hollow-core fiber.

Multimode fibers recently emerged as compact minimally-invasive probes for high-resolution deep-tissue imaging. However, the commonly used silica fibers have a relatively low numerical aperture (NA) limiting the spatial resolution of a probe. On top of that, light propagation within the solid core generates auto-fluorescence and Raman background, which interferes with imaging. Here we propose to use a hollow-core fiber to solve these problems. We experimentally demonstrate spatial wavefront shaping at the multimode hollow-core fiber output with tunable high-NA. We demonstrate raster-scan and speckle-based compressive imaging through a multimode hollow-core fiber.

NIR-II Orthogonal Fluorescent Ratiometric Nanoprobe for In Situ Bioimaging of Carbon Monoxide.

Carbon monoxide (CO) functions as a significant endogenous cell signaling molecule and is strongly associated with many physiological and pathological processes. However, conventional fluorescence imaging in the visible and near-infrared (NIR) I regions suffers autofluorescence background and photon scattering, hindering the accurate detection of CO in vivo. In addition, the complexity of physiological environments leads to fluctuating fluorescence emission. To solve these problems, herein, the NIR-II fluorescent nanoprobe NP-Pd for in vivo ratiometric bioimaging of CO is developed. In the presence of CO, NP-Pd exhibits responsive enhancement in absorption at 808nm, which amplifies the fluorescence signal of down-conversion nanoparticles (DCNP) at 1060nm under 808nm excitation, while the fluorescence signal of DCNP at 1525nm under 980nm excitation remains unchanged and serves as an internal standard. Through this orthogonally ratiometric fluorescence strategy, accurate CO bioimaging and precise diagnosis of acute liver injury diseases are achieved in the mouse model experiments, providing a novel tool for the in vivo detection of CO-related diseases.

Multiplexed Shortwave Infrared Imaging Highlights Anatomical Structures in Mice

AbstractMultiplexed fluorescence in vivo imaging remains challenging due to the attenuation and scattering of visible and traditional near infrared (NIR‐I, 650–950 nm) wavelengths. Fluorescence imaging using shortwave infrared (SWIR, 1000–1700 nm, a.k.a. NIR‐II) light enables deeper tissue penetration due to reduced tissue scattering as well as minimal background autofluorescence. SWIR‐emitting semiconductor quantum dots (QDs) with tunable emission peaks and optical stability are powerful contrast agents, yet few imaging demonstrations exclusively use SWIR emission beyond two‐color imaging schemes. In this study, we engineered three high quality lead sulfide/cadmium sulfide (PbS/CdS) core/shell QDs with distinct SWIR emission peaks ranging from 1100–1550 nm for simultaneous three‐color imaging in mice. We first use the exceptional photostability of QDs to non‐invasively track lymphatic drainage with longitudinal imaging, highlighting the detailed networks of lymphatic vessels with widefield imaging over a 2 hr period. We then perform multiplexed imaging with all three QDs to distinctly visualize the lymphatic system and spatially overlapping vasculature networks, including clearly distinguishing the liver and spleen. This work establishes optimized SWIR QDs for next generation multiplexed and longitudinal preclinical imaging, unlocking numerous opportunities for preclinical studies of disease progression, drug biodistribution, and cell trafficking dynamics in living organisms.

PH-responsive persistent luminescence nanoprobes biosensor for autofluorescence-free determination of glucose level in human samples and fingerprint encryption.

Glucose level is an important indicator of diabetes, and maintaining an appropriate physiological concentration of glucose is important for human health. However, traditional optical sensors are interfered by the interference of strong background autofluorescence and natural responsive luminescence, which severely limits their application in complex biological samples. Herein, as a novel glucose biosensing probe, green-emitting Zn2GeO4:Mn2+, Eu3+ (ZGME) persistent luminescence nanoparticles (PLNPs) with pH stimulus-responsive was prepared by a facile one-pot hydrothermal method. We also investigated the pH stimulus-responsive luminescence behaviour of ZGME over a range of pH values from 2.8 to 8.0. Taking advantage of the interesting property that ZGME photoluminescence intensity has a pH response, within an extraordinarily narrow pH range of 5.0-6.5 for highly selectivity and sensitive determination of glucose level in human samples by acid-responsive quenching and persistent luminescent performance. The detection results show high accuracy of the measured values of glucose in serum with a wide detection range (2.5μg L-1-10mg L-1) and low detection limit (0.5μg L-1). Finally, the pH-responsive persistent luminescence also makes ZGME promising for high-level fingerprint information encryption. Hence, the established pH stimulation-responsive PLNPs-based biosensing probe offers excellent performance with high selective, accuracy and signal-to-noise ratio for detection of glucose level in human samples.

Organelle-Targeting Polymer Dots Exhibiting Thermally Activated Delayed Fluorescence for Subcellular Imaging.

Selective imaging of specific subcellular structures provides valuable information about the cellular microenvironment. Materials exhibiting thermally activated delayed fluorescence (TADF) are rapidly emerging as metal-free probes with long-lived emission for intracellular time-gated imaging applications. Polymers incorporating TADF emitters can self-assemble into luminescent nanoparticles, termed polymer dots (Pdots), and this strategy enables them to circumvent the limitations of commercial organelle trackers and small molecule TADF emitters. In this study, diblock copolymers comprised of a hydrophilic block containing organelle-targeting monomers and a hydrophobic TADF-active block were synthesized by ring-opening metathesis polymerization (ROMP). Oxanorbornene-based monomers incorporating morpholine and triphenylphosphonium groups for lysosome and mitochondria targeting, respectively, were also synthesized. ROMP by sequential addition yielded well-defined diblock copolymers with dispersities <1.28. To analyze the effect of tuning the hydrophilic corona on cellular viability and uptake, we prepared Pdots with poly(ethylene glycol) (PEG) and bis-guanidinium (BGN) coronas, resulting in limited and efficient cellular uptake, respectively. Red-emissive Pdots with BGN-based coronas and organelle-targeting functionality were obtained with quantum yields up to 12% in water under air. Colocalization analysis confirmed that lysosome and mitochondria labeling in live HeLa cells was accomplished within 2 h of incubation, affording Pearson's correlation coefficients of 0.37 and 0.70, respectively. The potential application of these Pdots for time-resolved imaging is highlighted by a proof of concept using time-gated spectroscopy, which effectively separates the delayed emission of the TADF Pdots from the background autofluorescence of biological serum.

Multiplexed Shortwave Infrared Imaging Highlights Anatomical Structures in Mice.

Multiplexed fluorescence in vivo imaging remains challenging due to the attenuation and scattering of visible and traditional near infrared (NIR-I, 650-950 nm) wavelengths. Fluorescence imaging using shortwave infrared (SWIR, 1000-1700 nm, a.k.a. NIR-II) light enables deeper tissue penetration due to reduced tissue scattering as well as minimal background autofluorescence. SWIR-emitting semiconductor quantum dots (QDs) with tunable emission peaks and optical stability are powerful contrast agents, yet few imaging demonstrations exclusively use SWIR emission beyond two-color imaging schemes. In this study, we engineered three high quality lead sulfide/cadmium sulfide (PbS/CdS) core/shell QDs with distinct SWIR emission peaks ranging from 1100-1550 nm for simultaneous three-color imaging in mice. We first use the exceptional photostability of QDs to non-invasively track lymphatic drainage with longitudinal imaging, highlighting the detailed networks of lymphatic vessels with widefield imaging over a 2 hr period. We then perform multiplexed imaging with all three QDs to distinctly visualize the lymphatic system and spatially overlapping vasculature networks, including clearly distinguishing the liver and spleen. This work establishes optimized SWIR QDs for next generation multiplexed and longitudinal preclinical imaging, unlocking numerous opportunities for preclinical studies of disease progression, drug biodistribution, and cell trafficking dynamics in living organisms.

Persistent Luminescence Nanoplatform for Autofluorescence-Free Tracking of Submicrometer Plastic Particles in Plant.

The uptake of plastic particles by plants and their transport through the food chain make great risks to biota and human health. Therefore, it is important to trace plastic particles in the plant. Traditional fluorescence imaging in plants usually suffers significant autofluorescence background. Here, we report a persistent luminescence nanoplatform for autofluorescence-free imaging and quantitation of submicrometer plastic particles in plant. The nanoplatform was fabricated by doping persistent luminescence nanoparticles (PLNPs) onto polystyrene (PS) nanoparticles. Cr3+-doped zinc gallate PLNP was employed as the dopant for autofluorescence-free imaging due to its persistent luminescence nature. In addition, the Ga element in PLNP was used as a proxy to quantify the PS in the plant by inductively coupled plasma mass spectrometry (ICP-MS). Thus, the developed nanoplatform allows not only dual-mode autofluorescence-free imaging (persistent luminescence and laser-ablation ICP-MS) but also ICP-MS quantitation for tracking PS in plant. Application of this nanoplatform in a typical plant model Arabidopsis thaliana revealed that PS mainly distributed in the root (>99.45%) and translocated very limited (<0.55%) to the shoot. The developed nanoplatform has great potential for quantitative tracing of submicrometer plastic particles to investigate the environmental process and impact of plastic particles.

The pros and cons of nucleic acid-amplified immunoassays—a comparative study on the quantitation of prostate-specific antigen with and without rolling circle amplification

Integrating isothermal nucleic acid amplification strategies into immunoassays can significantly decrease analytical limits of detection (LODs). On the other hand, an amplification step adds time, complication, reagents, and costs to the assay format. To evaluate the pros and cons in the context of heterogeneous multistep immunoassays, we quantified prostate-specific antigen (PSA) with and without rolling circle amplification (RCA). In addition, we compared time-gated (TG) with continuous-wave (CW) photoluminescence (PL) detection using a terbium complex and a fluorescein dye, respectively. For both direct (non-amplified) and amplified assays, TG PL detection provided circa four- to eightfold lower LODs, illustrating the importance of autofluorescence background suppression even for multi-wash assay formats. Amplified assays required an approximately 2.4 h longer assay time but led to almost 100-fold lower LODs down to 1.3 pg/mL of PSA. Implementation of TG-FRET (using a Tb-Cy5.5 donor–acceptor pair) into the RCA immunoassay resulted in a slightly higher LOD (3.0 pg/mL), but the ratiometric detection format provided important benefits, such as higher reproducibility, lower standard deviations, and multiplexing capability. Overall, our direct comparison demonstrated the importance of biological background suppression even in heterogeneous assays and the potential of using isothermal RCA for strongly decreasing analytical LODs, making such assays viable alternatives to conventional enzyme-linked immunosorbent assays (ELISAs).Graphical