Simultaneous Fluorescence Detection Research Articles

A novel Psidium guajava and Cerium-MOF based dual-responsive bio-composite for the synchronous adsorptive removal and fluorescence detection of tetracycline antibiotics

The apprehensions about water contamination conduced by tetracycline antibiotics have raised serious concerns in scientific community worldwide. The researchers are intensively looking for materials that are sustainable and extend dual applicability in removal as well as detection of these contaminants. However, in this pursuit, fabrication of novel materials is still a great challenge. In this vein, inspired by the prodigious union of MOF and biobased materials, as enunciated by literature reports, present study unveils the fabrication of a novel dual responsive Psidium guajava and Cerium-MOF based bio-composite for simultaneous adsorption and fluorescence detection of tetracycline antibiotics. The fabricated Ce-MOF/0.5-GLP composite displayed excellent adsorption potential for removal of TCs within a very short initial time duration and rendered high adsorption capacities i.e. 81.30, 75.18 and 63.69 mg g−1 for DC, MC and TC, respectively. Conjointly, the fabricated bio-based Ce-MOF/0.5-GLP composite was engaged for selective fluorescence detection of TCs. The sensor exhibited exquisite selectivity and sensitivity for detection of TCs with low detection limit values i.e. 94.3 nM, 71.4 nM and 74.2 nM for DC, MC and TC, respectively. In light of the aforementioned findings, the developed composite illustrates a workable and realistic method for dealing with TCs via removal and detection.

Simultaneous detection of breast cancer biomarkers circROBO1 and BRCA1 based on a CRISPR-Cas13a/Cas12a system

Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.

Zinc nitride quantum dots as an efficient probe for simultaneous fluorescence detection of Cu2+ and Mn2+ ions in water samples.

The exceptional ascending heights of graphene (carbon) and boron nitride nanostructures have invited scientists to explore metal nitride nanomaterials. Herein, Zn3N2 quantum dots (QDs) were prepared via a simple hydrothermal route from the reaction between zinc nitrate hexahydrate and ammonia solution that possess efficient strength towards sensing applications of metal ions (Cu2+ and Mn2+). The as-prepared Zn3N2 QDs show bright fluorescence, displaying an emission peak at 408nm upon excitation at 320nm, with a quantum yield (QY) of 29.56%. It was noticed that the fluorescence intensity of Zn3N2 QDs linearly decreases with the independent addition of Cu2+ and Mn2+ ions, displaying good linearity in the ranges 2.5-50µM and 0.05-5µM with detection limits of 21.77nM and of 63.82nM for Cu2+ and Mn2+ ions, respectively. Theprobe was successfully tested for quantifying Cu2+ and Mn2+ in real samples including river, canal, and tap water, providinggood recoveries with a relative standard deviation < 2%. Furthermore, the masking proposition can successfully eliminate the interference if the twometal ions exist together. It was found that thiourea is efficiently able to mask Cu2+ and selectively quenches Mn2+, and L-cysteine is able to halt the quenching potential of Mn2+ and is selectively able to sense Cu2+. The Zn3N2 QDs provide a simple way for the simultaneous detection of both Cu2+ and Mn2+ ions in environmental samples at lowsample preparationsrequirements.

Dual-mode transfer response based on electrochemical and fluorescence signals for the detection of amyloid-beta oligomers (AβO).

An aptamer sensor has been developedutilizing a dual-mode and stimuli-responsive strategy for quantitative detection of AβO (amyloid-beta oligomers) through simultaneous electrochemical and fluorescence detection. To achieve this, we employed UIO-66-NH2 as a carrier container to load MB (Methylene Blue), and Fe3O4 MNPs (iron oxide magnetic nanoparticles) with aptamer (ssDNA-Fe3O4 MNPs) fixed on their surface for biological gating. The ssDNA-Fe3O4 MNPs were immobilized onto the surface of UIO-66-NH2 through hydrogen bonding between the aptamer and the -NH2 group on the surface of UIO-66-NH2, thereby encapsulating MB and forming ssDNA-Fe3O4@MB@UIO-66-NH2. During the detection of AβO, the aptamer selectively reacted with AβO to form the AβO-ssDNA-Fe3O4 complex, leading to its detachment from the surface of UIO-66-NH2. This detachment facilitated the release of MB, enabling its electrochemical detection. Simultaneously, the AβO-ssDNA-Fe3O4 complex was efficiently collected and separated using a magnet after leaving the container's surface. Furthermore, the addition of NaOH facilitated the disconnection of biotin modifications at the 3' end of the aptamer from the avidin modifications on the Fe3O4 MNPs. Consequently, the aptamer detached from the surface of Fe3O4 MNPs, resulting in the restoration of fluorescence intensity of FAM (fluorescein-5'-carboxamidite) modified at its 5' end for fluorescence detection. The dual-mode sensor exhibited significantly enhanced differential pulse voltammetry signals and fluorescence intensity compared to those in the absence of AβO. The sensor demonstrated a wide detection range of 10 fM to 10 μM, with a detection limit of 3.4 fM. It displayed excellent performance in detecting actual samples and holds promising prospects for early diagnosis of Alzheimer's disease.

Flexible Stacked Perovskite Photodetectors for High-Efficiency Multicolor Fluorescence Detection.

A flexible, multicolor detector based on stacked perovskite layers with graded band gaps was presented. Different perovskite layers generate a series of photocurrents corresponding to light intensities at different wavelengths. Experimentally, the flexible detector demonstrated acceptable long-term stability and temperature stability in the bending state. To demonstrate the advantages of the flexible multicolor detector in biological applications, a tubular-shaped multicolor fluorescence detector that embraces the sample cell was constructed. As a result, the detection limits of three kinds of CdTe quantum dots (QDs) with central wavelengths of 545, 625, and 730 nm were 0.52, 0.85, and 0.43 nM, respectively, which was significantly improved by more than 10 times compared to those of planar detectors. Additionally, the detector was able to detect three kinds of QDs simultaneously in a mixed solution, and the relative deviation was smaller than 10% compared to the preset concentration. These results demonstrate that the flexible stacked perovskite detector and the tubular-shaped detection configuration hold promise for the simultaneous fluorescent detection of multiple biomolecules.

Photoactive immunoconjugates for targeted photodynamic therapy of cancer.

Photodynamic therapy (PDT) has been used as an alternative or as a complement of conventional approaches for cancer treatment. In PDT, the reactive oxygen species (ROS) produced from the interaction between the photosensitizer (PS), visible light and molecular oxygen, kill malignant cells by triggering a cascade of cytotoxic reactions. In this process, the PS plays an extremely important role in the effectiveness of the therapy. In the present work, a new photoimmunoconjugate (PIC), based on cetuximab and the known third generation PS-glycophthalocyanine ZnPcGal4, was synthesized via reductive amination. The rationale behind this was the simultaneous cancer-associated specific targeting of PIC and photosensitization of targeted receptor positive cells. Varied reaction parameters and photodynamic conditions, such as PS concentrations and both type and intensities of light, were optimized. ZnPcGal4 showed significant photoactivity against EGFR expressing A431, EGFR-transfected HCT116 and HT29 cells when irradiated with white light of stronger intensity (38mW/cm2). Similarly, the synthesized PICs-T1 and T2 also demonstrated photoactivity with high intensity white light. The best optimized PIC: sample 28 showed no precipitation and aggregation when inspected visually and analyzed through SE-HPLC. Fluorescence excitation of sample 28 and 125I-sample 28 radioconjugate (125I-PIC, 125I-radiolabeling yield ≥95%, determined with ITLC) at 660nm showed presence of appended ZnPcGal4. In addition, simultaneous fluorescence and radioactivity detection of the 125I-PIC in serum and PBS (pH7.4) for the longest incubated time point of 72h, respectively, and superimposed signals thereof demonstrated ≥99% of loading and/or labeling yield, assuring overall stability of the PIC and corresponding PIC-radioconjugate w.r.t. both the appended ZnPcGal4 and bound-125I. Moreover, real-time binding analyses on EGFR-transfected HCT116 cells showed specific binding of 125I-PIC, suggesting no alternation in the binding kinetics of the mAb after appending it with ZnPcGal4. These results suggest dual potential applications of synthesized PICs both for PDT and radio-immunotherapy of cancer.

Nanoarchitectonics-Assisted Simultaneous Fluorescence Detection of Urinary Dual miRNAs for Noninvasive Diagnosis of Prostate Cancer.

Herein, we report a fluorescence strategy for the homogeneous and simultaneous analysis of urine miRNA-375 and miRNA-148a. The target miRNAs in urine bonded the devised dumbbell-shaped "C-Ag+-C" and "T-Hg2+-T" hairpin structures that could trigger cascade enzyme-free amplification. Then, the fluorescent CdTe quantum dots (QDs) and carbon dots (CDs) could selectively recognize Ag+ and Hg2+, to quantify the dual miRNAs concurrently. Under optimized conditions, the linear range was from 0.1 to 1000 fM and the limits of detection (LOD) for dual miRNAs reached 30 and 25 aM, respectively. The practicality was further evaluated with 45 clinical urine samples including prostate cancer (PC) and other patients, and the results were consistent with the clinical polymerase chain reaction (PCR) kit and ultrasonic and pathological findings. The receiver operating characteristic (ROC) curve analysis showed that the estimates of the area under the curve (AUC) were 0.739 for the serum prostate-specific antigen (PSA) and 0.941 for miRNA-375 and 0.946 for miRNA-148a. The sensitivity and specificity reached 75 and 100% for miRNA-375 and 71 and 94% for miRNA-148a, respectively, which was better than serum PSA. This strategy constructed a reliable system for dual miRNA detection in urine samples and proposed new insights into the rapid and noninvasive diagnosis of PC.

Spatial Confinement of Single-Drop System to Enhance Aggregation-Induced Emission for Detection of MicroRNAs

Due to high incidence, poor prognosis, and easy transformation into pancreatic cancer (PC) with high mortality, early diagnosis and prevention of acute pancreatitis (AP) have become significant research focuses. In this work, we proposed a magnetic single-drop microextraction (SDME) system with spatial confinement to enhance the aggregation-induced emission (AIE) effect for simultaneous fluorescence detection of miRNA-155 (associated with AP) and miRNA-196a (associated with PC). The target miRNAs were selectively recognized by the hairpin probe and triggered the DNA amplification reaction; then, the DNA strands with two independent probes of G-quadruplex/TAIN and Cy5 were constructed on the surfaces of the magnetic beads. The SDME process, in which a drop containing the fluorescence probes was formed at the tip of the magnetic microextraction rod rapidly within 10 s, was performed by magnetic extraction. In this way, G-quadruplex/TAIN was enriched owing to the spatial confinement of the single-drop system, and the fluorescence signal given off (by G-quadruplex/TAIN) was highly enhanced (AIE effect). This was detected directly by fluorescence spectrophotometry. The approach achieved low limits of detection of 2.1 aM for miRNA-196a and 8.1 aM for miRNA-155 and wide linear ranges from 10 aM to 10 nM for miRNA-196a and from 25 aM to 10 nM for miRNA-155. This novel method was applied to the fluorescence detection of miRNAs in human serum samples. High relative recoveries from 95.6% to 104.8% were obtained.

Hg(II) immobilization and detection using gel formation with tetra-(4-pyridylphenyl)ethylene and an aggregation-induced luminescence effect

Tetra-(4-pyridylphenyl)ethylene (TPPE), featuring an aggregation-induced luminescence effect (AIE), has been synthesized and used for selective detection of Hg2+ in DMF/H2O (3:7, v/v) binary solutions. There was a color change from colorless to yellow in the detection of the Hg2+ ions, in addition to an increased fluorescence emission. This shows that TPPE will function as an excellent “turn-on” fluorescence probe in the detection Hg2+. Moreover, the interference of Al3+, Ba2+, Mn2+, Ca2+, Fe3+, Cu2+, Ag+, Cd2+, Co2+, Ni2+, Mg2+, Pb2+, Zn2+, and Cr3+ ions was found to be negligible under optimized solvent conditions. Cysteine and EDTA were also found to form TPPE-based fluorescent switches with the Hg2+ ions. The practical use of the TPPE sensor was also demonstrated by using a specific test kit. Characterization using FT-IR, NMR titration, UV titration, EDS, and HR-MS techniques showed that Hg2+ will form a 1:1 complex with TPPE. Also, the observation of a Tyndall effect, in addition to UV absorption and fluorescence spectra, did clearly demonstrate the presence of an AIE. More noteworthy, TPPE and Hg2+ were found to form a metal–organic gel (MOG) in the DMF solution. The SEM, TEM, ICP, and Zeta potential analyses confirmed that the fluorescent MOG could further adsorb an excess of Hg2+ ions. The BET analyses revealed that the MOG showed a type IV-H3 hysteresis loop according to the International Union of Pure and Applied Chemistry classification. The results of the XRD analysis and of the spectroscopic titrations show that a π–π stacking may be the auxiliary driving force for the gel formation, after that a coordination has taken place. These results indicate that further research on structurally simple metal ion fluorescent probes, which are based on the AIE, is promising for the achievement of a simultaneous fluorescent detection and adsorption of heavy metal pollutants.

Photosynthetic Fluorescence from Earthlike Planets around Sunlike and Cool Stars

Remote sensing of the Earth has demonstrated that photosynthesis is traceable as the vegetation red edge (VRE), which is a steep rise in the reflection spectrum of vegetation, and as solar-induced fluorescence. This study examines the detectability of biological fluorescence from two types of photosynthetic pigments, chlorophylls (Chls) and bacteriochlorophylls (BChls), on Earthlike planets with oxygen-rich/poor and anoxic atmospheres around the Sun and M dwarfs. Atmospheric absorption, such as H2O, CH4, O2, and O3, and the VRE obscure the fluorescence emissions from Chls and BChls. We find that the BChl-based fluorescence for wavelengths of 1000–1100 nm, assuming the spectrum of BChl b–bearing purple bacteria, could provide a suitable biosignature, but only in the absence of water cloud coverage or other strong absorbers near 1000 nm. The Chl fluorescence is weaker for several reasons, e.g., spectral blending with the VRE. The apparent reflectance excess is greatly increased in both the Chl and BChl cases around TRAPPIST-1, due to the fluorescence and stellar absorption lines. This could be a promising feature for detecting the fluorescence around ultracool red dwarfs using follow-up ground-based observations at high spectral resolution; however, this would require a long time around Sunlike stars, even for a LUVOIR-like space mission. Moreover, the simultaneous detection of fluorescence and the VRE is the key to identifying traces of photosynthesis, because absorption, reflectance, and fluorescence are physically connected. For further validation of the fluorescence detection, the nonlinear response of biological fluorescence as a function of light intensity could be considered.

Simultaneous adsorption and fluorescent detection of Cr(VI) via lanthanide coordinating polymeric porous microparticles

Functional porous microparticles that enable simultaneous adsorption removal and online monitoring of hazardous hexavalent chromium are of great importance for effective remediation of Cr(VI) pollution. Herein, we have synthesized, for the first time, an oligomer bearing aromatic backbone and pendent carboxyl, sulfonate as well as amidoxime groups, which has been further employed as the dual functional ligand to coordinate with lanthanide ions of Tb3+ via a facile one-step solvent exchange induced self-assembling, leading to generation of the cube-like fluorescent porous microparticles. Thanks to their porous structure, abundant surface binding sites as well as green fluorescence emission that can be selectively quenched by Cr(VI), the obtained lanthanide coordinated polymeric porous microparticles exhibited both competitive adsorption and fluorescent detection of Cr(VI). More specifically, the optimized fluorescent porous microparticles demonstrated a maximum Cr(VI) adsorption capacity of 553.92 mg·g−1 and a limit of detection for Cr(VI) down to 1.92 nM according to systematic adsorption and detection experiments. On account of scalable synthesis of oligomer ligand, facile preparation of microparticles as well as their competitive dual adsorption and detection of Cr(VI), the current work basically opens new way for design and fabrication of advanced materials for heavy metal ions pollution controls.

Gold nanoclusters-engineered dual-emitting nanofibrous film for fluorescent discrimination and visual sensing of explosives

Development of reliable sensors for explosive detection is important to reduce explosion-related safety risks and mitigate widespread environmental concerns. However, it remains highly challenging to achieve on-site detection of multiple analytes in a simple yet efficient way. Herein, a flexible electrospun film-based sensing platform by engineering with dual-emitting gold nanoclusters (AuNCs) has been fabricated for the simultaneous fluorescent detection of two representative explosives, hexanitrohexaazaisowurtzitane (CL-20) and trinitrotoluene (TNT). Particularly, two types of AuNCs with different emission colors (green and red) and surface chemistry are designed, which can specifically respond to trace amounts of two explosives separately. While the presence of CL-20 can remarkably enhance the green emission of l-arginine/6-aza-2-thiothymine-protected AuNCs, the presence of TNT can efficiently quench the red emission of glutathione-protected AuNCs. Consequently, ratiometric fluorescent sensing of CL-20 and TNT can be achieved with these dual-emitting AuNCs (G/R-AuNCs), which exhibits a detection limit down to 0.55 and 2.38 µM, respectively. The distinct “turn on” and “turn off” response patterns of G/R-AuNCs towards two explosives could be harnessed to construct fluorescent “BUF” and “NOT” logic gates. Moreover, the present AuNCs-based explosive sensors were deposited into flexible electrospun nanofibrous film for visual and quantitative detection of CL-20 and TNT, which exhibit good mechanical property and promising sensing performance. This work provides a new approach to design explosive sensing platform by coupling the attractive features of fluorescent AuNCs to the electrospun nanofibrous film, which holds great potential for further practical applications.

Facile fluorescent probe for simultaneous fluorescence detection of fluoroquinolones and copper

Facile fluorescent probe for simultaneous fluorescence detection of fluoroquinolones and copper

A self-assembling split aptamer multiplex assay for SARS-COVID19 and miniaturization of a malachite green DNA-based aptamer

Multiplex assays often rely on expensive sensors incorporating covalently linked fluorescent dyes. Herein, we developed a self-assembling aptamer-based multiplex assay. This multiplex approach utilizes a previously established split aptamer sensor in conjugation with a novel split aptamer sensor based upon a malachite green DNA aptamer. This system was capable of simultaneous fluorescent detection of two SARS COVID-19-related sequences in one sample with individual sensors that possesses a limit of detection (LOD) in the low nM range. Optimization of the Split Malachite Green (SMG) sensor yielded a minimized aptamer construct, Mini-MG, capable of inducing fluorescence of malachite green in both a DNA hairpin and sensor format.

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry.

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter. Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels. Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter. Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels. Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

Investigation of the chemical effect of pilot injection on main combustion in a gasoline controlled auto-ignition engine by in-cylinder measurements and numerical simulation of H2O2, HO2, and OH radicals

Compared to conventional combustion in gasoline engines, Gasoline Controlled Auto-Ignition (GCAI) has the potential to significantly reduce both fuel consumption and nitrogen oxides (NOX) raw emissions. However, GCAI combustion still has a limited operating range due to limitations in combustion stability at low loads and high rates of cylinder pressure rise at high engine loads. Previous research identified hydrogen peroxide (H2O2) decomposition as an effective means to control GCAI, which is formed among other species with pilot fuel injection during the negative valve overlap (NVO) phase. Since the chemical effect of H2O2 on the main combustion has not been experimentally confirmed so far, the aim of the experimental study described in this paper is to detect H2O2 during early compression using optical methods. This is approached by conducting Photofragmentation Laser-Induced Fluorescence (PFLIF) of H2O2 by a pump laser and simultaneous hydroxyl radical Laser-Induced Fluorescence (OH-LIF) detection in the combustion chamber of a dedicated GCAI single-cylinder research engine at low loads after NVO with pilot injection (PI). Thereto, the pilot injection strategy as well as the global air/fuel ratio (AFR) are varied. Interestingly, no H2O2, hydroxyl (OH) or hydroperoxyl (HO2) constituents were detected under the operating conditions studied, despite relatively high pilot injection fuel masses and a highly accurate H2O2 detection limit of about 1 ppm. Numerical three-dimensional computational fluid dynamics (3D-CFD) combustion simulations using a semi-detailed reaction kinetic mechanism predicted 5–8 times higher H2O2 concentrations than the detection limit, yielding an uncertainty estimate of the numerical methods. Furthermore, the 3D-CFD simulations showed a significant mixture stratification which is a possible reason for the lack of H2O2 detection under these operating conditions.

Simultaneous Homogeneous Fluorescence Detection of AFP and GPC3 in Hepatocellular Carcinoma Clinical Samples Assisted by Enzyme-Free Catalytic Hairpin Assembly.

Simultaneous sensitive and cost-effective detection of multiple tumor markers has shown great potential for cancer diagnostics. Herein, we reported a simple enzyme-free parallel catalytic hairpin assembly (CHA) amplification strategy with N-methyl mesoporphyrin IX (NMM) and quantum dots (QDs) as signal reporters for the homogeneous fluorescent simultaneous detection of alpha-fetoprotein (AFP) and glypican-3 (GPC3). Upon selective binding, the released single-stranded DNA (ssDNA) from the two-aptamer double-stranded DNA (dsDNA) probes triggers CHA amplification, further releasing the G-quadruplex sequence and Ag+ from the C-Ag+-C structures at the same time. Then, NMM and CdTe QDs selectively recognize G-quadruplex and Ag+, respectively. Under optimized conditions, limits of detections (LODs) as low as 3 fg/mL for AFP and 0.25 fg/mL for GPC3 were achieved using fluorescence readout. Using color- and distance-based visual readouts, an LOD of 1 fg/mL for GPC3 was reached. This method was applied to quantitatively analyze AFP and GPC3 in 41 clinical serum samples of hepatocellular carcinoma (HCC) patients. The quantitative test results for AFP and GPC3 were consistent with those obtained using the electrochemiluminescence immunoassay (ECL-IA) clinical kit and correlated with radiological and pathological findings. The results of clinical tests demonstrated the potential of GPC3 as a tumor biomarker, and we propose a cut-off value of 2 ng/mL GPC3 for HCC.

Fluorescence lifetime-based pH mapping of tumors in vivo using genetically encoded sensor SypHerRed

Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications.

An Approach to the Simultaneous Detection of Multiple Biomarkers for the Early Diagnosis of Liver Cancer Using Quantum Dot Nanoprobes

Abstract Biomarker-based early diagnosis of liver cancer is of high clinical value for reducing the mortality rate. However, it has been challenging to establish early detection methods with a single biomarker such as alpha-fetoprotein (AFP) because of limited diagnostic sensitivity and specificity. Therefore, developing multiplexed biomarker detection assays is crucially important for early diagnosis. Yet, simultaneous detection methods involving three or more biomarkers have been scarce. Here we suggest employing the serological biomarker panel of glypican-3 (GPC3), dickkopf-1 (DKK1), and AFP for liver cancer detection. We present a rapid simultaneous detection approach for the biomarker panel labeled with three fluorescent quantum dot nanoprobes (emission wavelengths at 565 nm, 605 nm, and 655 nm). As a proof-of-concept, simultaneous fluorescence detection of the biomarker panel was demonstrated using mixed reference samples containing human recombinant GPC3, DKK1, and AFP antigens. Our simultaneous detection approach conferred a linear range of 0.625-2.5 ng•mL-1 for the entire biomarker panel, which merits further clinical validation for the simultaneous and accurate determination of the biomarker panel in human serum samples.