Tunneling nanotubes mediate KRas transport: Inducing tumor heterogeneity and altering cellular membrane mechanical properties.

Mutation in oncogene KRas plays a crucial role in the occurrence and progression of numerous malignant tumors. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. We hypothesize that oncogene KRas mutations are intrinsic to alterations in cellular mechanics that promote malignant tumor generation and progression. Here, we demonstrate the use of optical tweezers coupled with a confocal fluorescence imaging system and gene interference technique to reveal that the mutant KRas protein can be transported between homogeneous and heterogeneous tumor cells by tunneling nanotubes (TNTs), resulting in a significant reduction of membrane tension and acceleration of membrane phospholipid flow in the recipient cells. Simultaneously, the changes in membrane mechanical properties of the tumor cells also enhance the metastatic and invasive ability of the tumors, which further contribute to the deterioration of the tumors. This finding helps to clarify the association between oncogene mutations and changes in the mechanical properties of tumor cells, which provides a theoretical basis for the development of cancer treatment strategies. STATEMENT OF SIGNIFICANCE: Here, we present a laser confocal fluorescence system integrated with optical tweezers to observe the transfer of mutant KRasG12D protein from mutant cells to wild-type cells through TNTs. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. Our results demonstrate a significant decrease in membrane tension and an increase in membrane phospholipid flow in recipient cells. These alterations in mechanical properties augment the migration and invasive capabilities of tumor cells, contributing to tumor malignancy. Our findings propose that cellular mechanical properties could serve as new markers for tumor development, and targeting membrane tension may hold potential as a therapeutic strategy.

Intelligent near-infrared light-activatable DNA machine with DNA wire nano-scaffold-integrated fast domino-like driving amplification for high-performance imaging in live biological samples.

While there is significant potential for DNA machine-built enzyme-free fluorescence biosensors in the imaging analysis of live biological samples, they persist certain shortcomings. These encompass a deficiency of signal enrichment within a singular interface, uncontrolled premature activation during bio-delivery, and a slow reaction rate due to free nucleic acid collisions. In this contribution, we are committed to resolving the above challenges. Firstly, a single-interface-integrated domino-like driving amplification is constructed. In this conception, a specific target acts as the domino promotor (namely the energy source), initiating a cascading chain reaction that grafts onto a singular interface. Next, an 808 nm near-infrared (NIR) light-excited up-converting luminescence-induced light-activatable biosensing technique is introduced. By locking the target-specific identification segment with a photo-cleavage connector, the up-converted ultraviolet emission can activate target binding in a completely controlled manner. Moreover, a fast reaction rate is achieved by confining nucleic acid collisions within the surface of a DNA wire nano-scaffold, leading to a substantial enhancement in local contact concentration (30.8-fold increase, alongside a 15 times elevation in rate). When a non-coding microRNA (miRNA-221) is positioned as the model low-abundance target for proof-of-concept validation, our intelligent DNA machine demonstrates ultra-high sensitivity (with a limit of detection down to 62.65 fM) and good specificity for this hepatic malignant tumor-associated biomarker in solution detection. Going further, it is worth highlighting that the biosensing system can be employed to carry out high-performance imaging analysis in live bio-samples (ranging from the cellular level to the nude mouse body), thereby propelling the field of DNA machines in disease diagnosis.

Detection of free DNA based on metal-enhanced fluorescence triggered by CRISPR-Cas12a and colorimetric analysis.

The CRISPR-Cas system has been found to be extremely sensitive and there is an urgent demand to extend its potential in bioassays. Herein, we developed a novel nanobiosensor to detect the human papillomavirus 16 genes (HPV-16 DNA), which is triggered by CRISPR-Cas12a to amplify the fluorescence signal by metal-enhanced fluorescence (CAMEF). Along with the changing of the fluorescence signal, the aggregation of the substrate of MEF also leads to a change in the color of the mixture solution, enabling dual signal detection with the fluorescence and the naked eye. Furthermore, the designed CAMEF probe was verified to detect the HPV-16 DNA accurately and reliably in biological samples. Triggered by the CRISPR system, the designed CAMEF probe allows quantitative detection of the HPV-16 DNA in the wide range of 10-500 pM. Owing to the MEF, the fluorescence signal of the CAMEF probe was significantly amplified with the detection limit as low as 1 pM. Besides, we can determine the concentration of HPV-16 DNA simply by the naked eye, which also drastically reduces the possibility of false-positive signals. Theoretically, the target ssDNA could be any strand of DNA obtained by designing the crRNA sequence in the CRISPR-Cas system. We believe that the designed CAMEF sensor can present a reliable approach for the accurate detection of low amounts of target ssDNA in complex biological samples.

Near-Infrared Light-Powered and DNA Nanocage-Confined Catalytic Hairpin Assembly Nanobiosensor with a Nucleic Acid Restriction Behavior and Reinforced Enzymatic Resistance for Robust Imaging Assay in Live Biosystems.

While DNA amplifier-built nanobiosensors featuring a DNA polymerase-free catalytic hairpin assembly (CHA) reaction have shown promise in fluorescence imaging assays within live biosystems, challenges persist due to unsatisfactory precision stemming from premature activation, insufficient sensitivity arising from low reaction kinetics, and poor biostability caused by endonuclease degradation. In this research, we aim to tackle these issues. One aspect involves inserting an analyte-binding unit with a photoinduced cleavage bond to enable a light-powered notion. By utilizing 808 nm near-infrared (NIR) light-excited upconversion luminescence as the ultraviolet source, we achieve entirely a controllable sensing event during the biodelivery phase. Another aspect refers to confining the CHA reaction within the finite space of a DNA self-assembled nanocage. Besides the accelerated kinetics (up to 10-fold enhancement) resulting from the nucleic acid restriction behavior, the DNA nanocage further provides a 3D rigid skeleton to reinforce enzymatic resistance. After selecting a short noncoding microRNA (miRNA-21) as the modeled low-abundance sensing analyte, we have verified that the innovative NIR light-powered and DNA nanocage-confined CHA nanobiosensor possesses remarkably high sensitivity and specificity. More importantly, our sensing system demonstrates a robust imaging capability for this cancer-related universal biomarker in live cells and tumor-bearing mouse bodies, showcasing its potential applications in disease analysis.

Photoresponsive CHA-Integrated Self-Propelling 3D DNA Walking Amplifier within the Concentration Localization Effect of DNA Molecular Framework Enables Highly Efficient Fluorescence Bioimaging.

While three-dimensional (3D) DNA walking amplifiers hold considerable promise in the construction of advanced DNA-based fluorescent biosensors for bioimaging, they encounter certain difficulties such as inadequate sensitivity, premature activation, the need for exogenous propelling forces, and low reaction rates. In this contribution, a variety of profitable solutions have been explored. First, a catalytic hairpin assembly (CHA)-achieved nonenzymatic isothermal nucleic acid amplification is integrated to enhance sensitivity. Subsequently, one DNA component is simply functionalized with a photocleavage-bond to conduct a photoresponsive manner, whereby the target recognition occurs only when the biosensor is exposed to an external ultraviolet light source, overcoming premature activation during biodelivery. Furthermore, a special self-propelling walking mechanism is implemented by reducing biothiols to MnO2 nanosheets, thereby propelling forces that are self-supplied to a Mn2+-reliant DNAzyme. By carrying the biosensing system with a DNA molecular framework to induce a unique concentration localization effect, the nucleic acid contact reaction rate is notably elevated by 6 times. Following these, an ultrasensitive in vitro detection performance with a limit of detection down to 2.89 fM is verified for a cancer-correlated microRNA biomarker (miRNA-21). Of particular importance, our multiple concepts combined 3D DNA walking amplifier that enables highly efficient fluorescence bioimaging in live cells and even bodies, exhibiting a favorable application prospect in disease analysis.

Genetically engineered virus-like particle-armoured and multibranched DNA scaffold-corbelled ultra-sensitive hierarchical hybridization chain reaction for targeting-enhanced imaging in living biosystems under spatiotemporal light powering.

Although nucleic acids-based fluorescent biosensors, exemplified by the hybridization chain reaction (HCR), have exhibited promise as an imaging tool for detecting disease-related biomolecular makers in living biosystems, they still face certain challenges. These include the need for improved sensitivity, poor bio-targeting capability, the absence of signal enrichment interface and the uncontrollable biosensing initiation. Herein, we present a range of effective solutions. First, a stacking design resembling building blocks is used to construct a special hierarchical HCR (termed H-HCR), for which a hierarchical bridge is employed to graft multiunit HCR products. Furthermore, the H-HCR components are encapsulated into a virus-like particle (VLP) endowed with a naturally peptide-mediated targeting unit through genetic engineering of plasmids, after which the biosensor can specifically identify cancer cytomembranes. By further creating a multibranched DNA scaffold to enrich the H-HCR produced detection signals, the biosensor's analyte recognition module is inserted with a photocleavage-linker, allowing that the biosensing process can be spatiotemporally initiated via a light-powered behavior. Following these innovations, this genetically engineered VLP-armoured and multibranched DNA-scaffold-corbelled H-HCR demonstrates an ultra-sensitive and specific biosensing performance to a cancer-associated microRNA marker (miRNA-155). Beyond the worthy in vitro analysis, our method is also effective in performing imaging assays for such low-abundance analyte in living cells and even bodies, thus providing a roust platform for disease diagnosis.

Multiple miRNA Detection through a Suspended Microbead Array Encoded by Triple-Color Upconversion Luminescent Nanotags via Bi-Beam Splitter Hybrid-Multitrap Optical Tweezers.

In recent years, optical tweezers have become a novel tool for biodetection, and to improve the inefficiency of a single trap, the development of multitraps is required. Herein, we constructed a set of hybrid multitrap optical tweezers with the balance of stability and flexibility by the combination of two different beam splitters, a diffraction optical element (DOE) and galvano mirrors (GMs), to capture polystyrene (PS) microbeads in aqueous solutions to create an 18-trap suspended array. A sandwich hybridization strategy of DNA-miRNA-DNA was adopted to detect three kinds of target miRNAs associated with triple negative breast cancer (TNBC), in which different upconversion nanoparticles (UCNPs) with red, green, and blue emissions were applied as luminescent tags to encode the carrier PS microbeads to further indicate the levels of the targets. With encoded luminescent microbeads imaged by a three-channel microscopic system, the biodetection displayed high sensitivity with low limits of detection (LODs) of 0.27, 0.32, and 0.33 fM and exceptional linear ranges of 0.5 fM to 1 nM, 0.7 fM to 1 nM, and 1 fM to 1 nM for miR-343-3p, miR-155, and miR-199a-5p, respectively. In addition, this bead-based assay method was demonstrated to have the potential for being applied in patients' serum by satisfactory standard addition recovery experiment results.

A Photoresponsive and Metal-Organic Framework Encapsulated DNA Tetrahedral Entropy-Driven Amplifier for High-Performance Imaging Intracellular MicroRNA.

The further development of high-performance fluorescent biosensors to image intracellular microRNAs is beneficial to cancer medicine. By virtue of the need for enzymes and hairpin DNA probes, the entropy-driven reaction-assisted signal amplification strategy has shown an enormous potential to accomplish this task. Nevertheless, this good option still meets with poor biostability, low cell uptake efficiency, and unsatisfactory accuracy. On the basis of these challenges, we put forward here a battery of solving pathways. First, the straight DNA probes are anchored onto the vertexes of dual DNA tetrahedrons, and thus the enzyme resistance of the whole sensing system is observably enhanced. A metal-organic framework (ZIF-8 nanoparticle), which can be effectively dissociated into a weakly acidic environment, then is employed as an additional delivery vehicle to encapsulate such a DNA tetrahedron sustained biosensor and finally bring about a more efficient endocytosis. Last, a kind of photocleavage-linker triggered photoresponsive manner is incorporated to achieve an exceptional precise target identification, by which the biosensor can only be initiated under the irradiation of an externally mild 365 nm ultraviolet light source. In accordance with the above efforts, worthy assay performance toward microRNA-196a has given rise to this newly constructed biosensor, whose sensitivity is down to 2.7 pM and also able to distinguish single-base variation. Beyond that, the amplifier can work as a powerful imaging toolbox to accurately determine the targets in living cells, providing a promising intracellular sensing platform.

Biomimetic Chip Enhanced Time-Gated Luminescent CRISPR-Cas12a Biosensors under Functional DNA Regulation.

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

Photo-gated and self-powered three-dimensional DNA motors with boosted biostability for exceptionally precise and efficient tracing of intracellular survivin mRNA.

Benefiting from the outstanding signal amplification effect and the admirable construction flexibility, the currently proposed DNA motors (particularly DNA walkers) based biosensing concepts have provided a forceful fluorescence imaging tool for intracellular detection. Even so, this promising sensing means is not only subject to poor controllability and prone to produce false signals but also requires exogenous powering forces owing to the common employment of DNAzyme. In response to these challenges, we are herein motivated to present some meaningful solving strategies. For one thing, the surfaces of gold nanoparticles are conducted with a photo-gated walking behavior by introducing a photocleave mode, under which the light-switchable DNA walkers are capable of being selectively activated via an external ultraviolet source to faultlessly prevent the sensing frame from being pre-initiated during cellular uptake and intracellular delivery. For another, the intracellular biothiols are consumed by MnO2 nanosheets to effectively avoid the competitions to Au-S bonds to eliminate potential false outputs and also self-supply sufficient cofactors (Mn2+) to actualize a self-powered operation pattern as well as facilitate the endocytosis process. Following these breakthroughs, a favorable analysis performance towards a model tumor biomarker (survivin mRNA) is endowed with the newly raised biosensor, whose sensitivity is low to pM level with a sound specificity for identifying single base mismatching. Moreover, the significantly improved autonomous three-dimensional DNA walkers can be used to determine and dynamically trace the targets in live cancer cells with an exceptional precise and efficient manner, commendably impelling the sensing ability of DNA motors in biological specimens.

Light-Activated and Self-Driven Autonomous DNA Nanomachine Enabling Fluorescence Imaging of MicroRNA in Living Cells with Exceptional Precision and Efficiency.

Owing to their favorable design flexibility and eminent signal amplification ability, DNA nanomachine-supported biosensors have provided an attractive avenue for intracellular fluorescence imaging, especially for DNA walkers. However, this promising option not only suffers from poor controllability but also needs to be supplied with additional driving forces on account of the frequent employment of metal ion-dependent DNAzymes. Aiming at overcoming these obstacles, we introduce some fruitful solutions. On one hand, innovative light-activated walking behavior induced by a photocleavage mode is established on the surfaces of gold nanoparticles, and such a photoselective sensing system can be perfectly prevented from pre-activating during the intracellular delivery process and made to achieve target identification only under irradiation using a moderate ultraviolet light source. On the other hand, this light-switchable sensing frame is encapsulated within a dissociable metal-organic framework (ZIF-8) to facilitate endocytosis and ensure sufficient internal cofactors (Zn2+) to realize a self-driven pattern in the acidic environment of the cell lysosome. Based on the abovementioned efforts, the newly constructed autonomous three-dimensional DNA walkers present satisfactory sensitivity (a limit of detection of down to 19.4 pM) and specificity (even distinguishing single-base changes) toward a model biomarker (microRNA-21). More importantly, the sensing method allows determination of the variations in targets in living cancer cells with exceptional precision and efficiency, offering a powerful assay platform for intracellular imaging.

Spectrally Combined Encoding for Profiling Heterogeneous Circulating Tumor Cells Using a Multifunctional Nanosphere-Mediated Microfluidic Platform.

Comprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single-cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding (SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere-mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single-cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral-readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.

Mitigation of impact of a major benzene spill into a river through flow control and in-situ activated carbon absorption.

Benzene is a toxic contaminant and can harm many aquatic species and cause serious damages to the river eco-system, if released to rivers. In 2012, a major spill accident occurred on the Huaihe River in Eastern China with 3 tons of benzene released to the river section 70 km upstream of a natural reserve. Two emergency measures were taken to minimize the impact of the accident on the natural reserve: 1) flow control by adjusting upstream sluices to delay the arrival of the contaminant plume at the reserve and 2) in-situ treatment using activated carbons to reduce the contaminant concentration. Here we develop a process-based mathematical model to analyze the monitoring data collected shortly after the accident, and explore not only how effective the adopted measures were over the incident but more importantly the mechanisms and critical conditions underlying the effectiveness of these measures. The model can be used as a tool for designing optimal management responses to similar spill accidents in regulated river systems, combining flow control and in-situ treatment.

Bioinspired sensor chip for detection of miRNA-21 based on photonic crystals assisted cyclic enzymatic amplification method.

Cancer, as the most invasive disease in the world, has led to an increasing amount of death year by year, so it is highly desired to develop a portable device to monitor the aberrant expression of biomarker in cancer patient. Here, we present a bio-photonic periodic nanostructures sensor chip assisted cyclic enzymatic amplification method to detect miRNA-21 with a detection limit of 55 fM. By employing biocompatible polydopamine nanospheres (PDANs) and DNaseⅠto construct an target-recycling amplification process on the photonic crystals, the output fluorescence signal can be strengthened selectively and short amplification time is needed. Benefiting from the synergy of the enhancement of photonic crystals and enzymatic cycle amplification, we realize high sensitivity detection of miRNA-21 with a detection range of 1 pM-10 nM and a detection limit of about four orders of magnitudes lower than the method employs no amplification, showing an expectable prospect in the early diagnosis of cancer.

Real-Time Monitoring of Temperature Variations around a Gold Nanobipyramid Targeted Cancer Cell under Photothermal Heating by Actively Manipulating an Optically Trapped Luminescent Upconversion Microparticle.

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.

Breaking Through Bead-Supported Assay: Integration of Optical Tweezers Assisted Fluorescence Imaging and Luminescence Confined Upconversion Nanoparticles Triggered Luminescent Resonance Energy Transfer (LRET).

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

Combining Holographic Optical Tweezers with Upconversion Luminescence Encoding: Imaging-Based Stable Suspension Array for Sensitive Responding of Dual Cancer Biomarkers.

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

Target-triggered signal turn-on detection of prostate specific antigen based on metal-enhanced fluorescence of Ag@SiO2@SiO2-RuBpy composite nanoparticles.

A three-layer core-shell nanostructure consisting of a silver core, a silica spacer, and a fluorescent dye RuBpy-doped outer silica layer was fabricated, and the optimal metal-enhanced fluorescence (MEF) distance was explored through adjusting the thickness of the silica spacer. The results show that the optimal distance is ∼10.4 nm with the maximum fluorescence enhancement factor 2.12. Then a new target-triggered MEF 'turn-on' strategy based on the optimized composite nanoparticles was successfully constructed for quantitative detection of prostate specific antigen (PSA), by using RuBpy as the energy donor and BHQ-2 as the acceptor. The hybridization of the complementary DNA of PSA-aptamer immobilized on the surface of the MEF nanoparticles with PSA-aptamer modified with BHQ-2, brought BHQ-2 in close proximity to RuBpy-doped silica shell and resulted in the decrease of fluorescence. In the presence of target PSA molecules, the BHQ-PSA aptamer is dissociated from the surface of the nanoparticles with the fluorescence switched on. Therefore, the assay of PSA was achieved by measuring the varying fluorescence intensity. The results show that PSA can be detected in the range of 1-100 ng ml-1 with a detection limit of 0.20 ng ml-1 (6.1 pM), which is 6.7-fold increase of that using hollow RuBpy-doped silica nanoparticles. Moreover, satisfactory results were obtained when PSA was detected in 1% serum.

Metal-enhanced fluorescent dye-doped silica nanoparticles and magnetic separation: A sensitive platform for one-step fluorescence detection of prostate specific antigen.

The world health organization figures show prostate cancer in developed countries has been the second primary cause of cancer mortality following lung cancer for the men. So, early and sensitive diagnosis of cancer is very important before it spreads out to the other organs of the body. It is well-known that prostate-specific antigen (PSA) is the most specific and efficient tumor marker for the diagnosis of prostate cancer. Herein, we successfully fabricated core-shell composite fluorescent nanoparticle Ag@SiO2@SiO2-RuBpy which provide a photoluminescence enhancement of up to ~3-fold when the separation distance between the surface of silver core and the center of the third RuBpy doped silica shell is about 10nm. These core-shell MEF-capable nanoparticles have obvious advantages. The interaction between the doped RuBpy molecules in the outer silica layer and the silver core, greatly improves the excitation efficiency and enhances the fluorescence intensity. Importantly, the presence of silica can reduce the self-quenching of RuBpy, which makes larger amounts of RuBpy incorporated into the silica shell. In addition, the shell protects the RuBpy against collisional quenching and irreversible photodegradation and provides abundant hydroxyl for easy conjugation. After that a highly sensitive, specific and reliable strategy based on metal-enhanced fluorescence and magnetic separation was applied for the detection of PSA in both buffer and serum. The process could be rapidly accomplished, in which the immunomagnetic nanospheres (IMNs) and immunofluorescent nanoparticles (IFNs) were used to capture and identify the target molecules simultaneously. A good linear relationship between the fluorescence intensity and the concentration of PSA (0.1-100ng/mL) with a detection limit 27pg/mL was obtained.

One-step separation-free detection of carcinoembryonic antigen in whole serum: Combination of two-photon excitation fluorescence and optical trapping.

Direct analysis of biomolecules in complex biological samples remains a major challenge for fluorescence-based approaches due to the interference of background signals. Herein, we report an analytical methodology by exploiting a single low-cost near-infrared sub-nanosecond pulse laser to synchronously actualize optical trapping and two-photon excitation fluorescence for senstive detection of carcinoembryonic antigen (CEA) in buffer solution and human whole serum with no separation steps. The assay is performed by simultaneously trapping and exciting the same immune-conjugated microsphere fabricated with a sandwich immunization strategy. Since the signal is strictly limited in the region of a three-dimensional focal volume where the microsphere is trapped, no obvious background signal is found to contribute the detected signals and thus high signal-to-background data are obtained. As a proof-of-concept study, the constructed platform exhibits good specificity for CEA and the detection limit reaches as low as 8pg/mL (45 fM) with a wide linear range from 0.01 to 60ng/mL in the both cases. To investigate the potential application of this platform in clinical diagnosis, 15 cases of serum samples were analyzed with satisfactory results, which further confirm the applicability of this method.