Application of Spectral Crosstalk Correction for Improving Multiplexed MicroRNA Detection Using a Single Excitation Wavelength

MicroRNAs (miRNAs) play crucial roles in the regulation of cellular activities and are next-generation biomarkers for early cancer detection. Simultaneous monitoring of multiplexed miRNA is very important for enhancing the accuracy of cancer diagnostics. Traditional fluorescence methods for multicomponent analysis were usually operated under multiple excitation wavelengths, because spectral crosstalk is very detrimental to detecting accuracy for multicomponent analysis. Herein, we present a fluorescence strategy for multi-miRNAs detection in plasma under a single excitation wavelength. Nucleic acid stain TOTO-1 and three labeled fluorescence dyes Cy3, Cy3.5, and Cy5 emit no fluorescence in their free state. Target miRNA hybridized the auxiliary and probe oligonucleotides into duplex nucleic acid. Intercalation interaction localized TOTO-1 and labeled dyes into the duplex nucleic acid. As a result, TOTO-1 emitted strong fluorescence and efficient Förster resonance energy transfer (FRET) happened. MicroRNAs miRNA-155, miRNA-182, and miRNA-197, which are significant for the early diagnosis of lung cancer, were simultaneously detected as models. Deviations from spectral crosstalk in the presence of other miRNAs were corrected by mathematical methods. Results demonstrated that, after spectra crosstalk corrections, every miRNA at high or low concentration in plasma was determined accurately in the presence of either high or low concentrations of the other two miRNAs. This new multiplexed assay for miRNAs is promising for clinical diagnosis, prognosis, and therapeutic monitoring of early-stage lung cancer

Label-Free Detection of Telomerase Activity in Urine Using Telomerase-Responsive Porous Anodic Alumina Nanochannels

Telomerase is closely related to cancers, which makes it one of the most widely known tumor marker. Recently, many methods have been reported for telomerase activity measurement in which complex label procedures were commonly used. In this paper, a label-free method for detection of telomerase activity in urine based on steric hindrance changes induced by confinement geometry in the porous anodic alumina (PAA) nanochannels was proposed. Telomerase substrate (TS) primer was first assembled on the inside wall of PAA nanochannels by Schiff reaction under mild conditions. Then, under the action of telomerase, TS primer was amplified and extended to repeating G-rich sequences (TTAGGG)_<i>x</i>, which formed multiplex G-quadruplex in the presence of potassium ions (K⁺). This configurational change led to the increment of steric hindrance in the nanochannels, resulting in the decrement of anodic current of potassium ferricyanide (K₃[Fe­(CN)₆]). Compared with previously reported methods based on PAA nanochannels (usually one G-quadruplex formed), multiplex repeating G-quadruplex formed on one TS primer in this work. As a result, large current drop (∼3.6 μA, 36%) was obtained, which gave facility to improve the detection sensitivity. The decreased ratio of anodic current has a linear correlation with the logarithm of HeLa cell number in the range of 10–5000 cells, with the detection limit of seven cells. The method is simple, reliable, and has been successfully applied in the detection of telomerase in urine with good accuracy, selectivity and reproducibility. In addition, the method is nondestructive test compared to blood analysis and pathology tests, which is significant for cancer discovery, development, and prognosis

Enhanced Enzymatic Reactivity for Electrochemically Driven Drug Metabolism by Confining Cytochrome P450 Enzyme in TiO₂ Nanotube Arrays

Understanding the enzymatic reaction kinetics that occur within a confined space or interface is a significant challenge. Herein, a nanotube array enzymatic reactor (CYP2C9/Au/TNA) was constructed by electrostatically adsorbing enzyme on the inner wall of TiO₂ nanotube arrays (TNAs). TNAs with different dimensions could be fabricated by the anodization of titanium foil through varying the anodization potential or time. The electrical conductivity of TNAs was improved by electrodepositing Au nanoparticles on the inner wall of TNAs. The cytochrome P450 2C9 enzyme (CYP2C9) was confined inside TNAs as a model. The enzymatic activity of CYP2C9 and tolbutamide metabolic yield could be effectively regulated by changing the nanotube diameter and length of TNAs. The enzymatic rate constant <i>k</i>_cat and apparent Michaelis constant <i>K</i>_m^app were determined to be 9.89 s^–1 and 4.8 μM at the tube inner diameter of about 64 nm and length of 1.08 μm. The highest metabolic yield of tolbutamide reached 14.6%. Furthermore, the designed nanotube array enzymatic reactor could be also used in situ to monitor the tolbutamide concentration with sensitivity of 28.8 μA mM^–1 and detection limit of 0.52 μM. Therefore, the proposed nanotube array enzymatic reactor was a good vessel for studying enzyme biocatalysis and drug metabolism, and has potential applications including efficient biosensors and bioreactors for chemical synthesis

Voltammetric Study and Modeling of the Electrochemical Oxidation Process and the Adsorption Effects of Luminol and Luminol Derivatives on Glassy Carbon Electrodes

Luminol is one of the most widely used electrochemiluminescence (ECL) reagents, yet the detailed mechanism and kinetics of the electrochemical oxidation of luminol remain unclear. We propose a model that describes the electrochemical oxidation of luminol as multiple electron transfer reactions followed by an irreversible chemical reaction, and we applied a finite element method simulation to analyze the electron transfer kinetics in alkaline solutions. Although negligible at higher pH values, the adsorption of luminol on the glassy carbon electrode became noticeable in a solution with pH = 12. Additionally, various types of adsorption behaviors were observed for luminol derivatives and analogues, indicating that the molecular structure affected not only the oxidation but also the adsorption process. The adsorption effect was analyzed through a model with a Langmuir isotherm to show that the saturated surface concentration as well as the reaction kinetics increased with decreasing pH, suggesting a competition for the active sites between the molecule and OH–. Moreover, we show that the ECL intensity could be boosted through the adsorption effect by collecting the ECL intensity generated through the electrochemical oxidation of luminol and a luminol analogue, L012, in a solution with pH = 13. In contrast with luminol, a significant adsorption effect was observed for L012 at pH = 13, and the ECL intensity was enhanced by the adsorbed species, especially at higher scan rates. The magnitude of the enhancement of the ECL intensity matched well with the simulation using our model

Construction of Three-Dimensional Hemin-Functionalized Graphene Hydrogel with High Mechanical Stability and Adsorption Capacity for Enhancing Photodegradation of Methylene Blue

A three-dimensional hemin-functionalized graphene hydrogel (Hem/GH) was prepared by a facile self-assembly approach. The as-prepared Hem/GH showed good mechanical strength with a storage modulus of 609–642 kPa and a high adsorption capacity to organic dye contaminants (341 mg g^–1 for rhodamine B). Moreover, Hem/GH could be used as a photosensitizer for the photocatalytic degradation of organic dyes and displayed superior photodegradation activity of methylene blue (MB). This result was better than that of counterparts such as graphene hydrogel (GH) and commercial catalyst P25. The excellent cycling performance of the Hem/GH was well maintained even after multiple cycles on adsorption process and photocatalytic reaction. Interestingly, after the photodegradation of MB, a light-induced pH change of the solution from alkaline pH 8.99 to acidic pH 3.82 was observed, and 10 wt % total organic carbon remained. The liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) analysis confirmed the generation of acidic degradation products. The photocatalytic mechanism was further investigated by trapping experiments, which revealed that the MB degradation was driven mainly by the participation of O₂^•– radicals in the photocatalytic reaction. As an extended application, visually intuitive observation showed the as-prepared Hem/GH also had strong antibacterial properties. These results suggest that Hem/GH could be potentially used for practical application due to its high adsorption ability, excellent photocatalytic activity, and strong antibacterial properties

Visual, Label-Free Telomerase Activity Monitor via Enzymatic Etching of Gold Nanorods

Early diagnosis and life-long surveillance are clinically important to improve the long-term survival of cancer patients. Telomerase activity is a valuable biomarker for cancer diagnosis, but its measurement often used complex label procedures. Herein, we designed a novel, simple, visual and label-free method for telomerase detection by using enzymatic etching of gold nanorods (GNRs). First, repeating (TTAGGG)_<i>x</i> sequences were extented on telomerase substrate (TS) primer. It formed G-quadruplex under the help of Hemin and K⁺. Second, the obtained horseradish peroxidase mimicking hemin/G-quadruplex catalyzed the H₂O₂-mediated etching of GNRs to the short GNRs, even to gold nanoparticles (GNPs), generating a series of distinct color changes due to their plasmon-related optical response. Thus, this enzymatic reaction can be easily coupled to telomerase activity, allowing for the detection of telomerase activity based on vivid colors. This can be differentiated sensitively by naked eyes because human eyes are more sensitive to color variations rather than the optical density variations. As a result, telomerase activity can be quantitatively detected ranging from 200 to 15000 HeLa cells mL^–1. The detection limit was 90 HeLa cells mL^–1 (<i>S</i>/<i>N</i> = 3). Importantly, the application of this method in bladder cancer samples was in agreement with the clinical results. Thus, this method was considerably suitable for point-of-care diagnostics in resource-constrained regions because of the easy readout of results without the use of sophisticated apparatus

Nanostructured 2D Diporphyrin Honeycomb Film: Photoelectrochemistry, Photodegradation, and Antibacterial Activity

Surface patterns of well-defined nanostructures play important roles in fabrication of optoelectronic devices and applications in catalysis and biology. In this paper, the diporphyrin honeycomb film, composed of titanium dioxide, protoporphyrin IX, and hemin (TiO₂/PPIX/Hem), was synthesized using a dewetting technique with the well-defined polystyrene (PS) monolayer as a template. The TiO₂/PPIX/Hem honeycomb film exhibited a higher photoelectrochemical response than that of TiO₂ or TiO₂/PPIX, which implied a high photoelectric conversion efficiency and a synergistic effect between the two kinds of porphyrins. The TiO₂/PPIX/Hem honeycomb film was also a good photosensitizer due to its ability to generate singlet oxygen (¹O₂) under irradiation by visible light. This led to the use of diporphyrin TiO₂/PPIX/Hem honeycomb film for the photocatalytic inactivation of bacteria. In addition, the photocatalytic activities of other metal-diporphyrin-based honeycomb films, such as TiO₂/MnPPIX/Hem, TiO₂/CoPPIX/Hem, TiO₂/NiPPIX/Hem, TiO₂/CuPPIX/Hem, and TiO₂/ZnPPIX/Hem, were investigated. The result demonstrated that the photoelectric properties of diporphyrin-based film could be effectively enhanced by further coupling of porphyrin with metal ions. Such enhanced performance of diporphyrin compounds opened a new way for potential applications in various photoelectrochemical devices and medical fields

Potential-Modulated Electrochemiluminescence of Carbon Nitride Nanosheets for Dual-Signal Sensing of Metal Ions

As an emerging semiconductor, graphite-phase polymeric carbon nitride (GPPCN) has drawn much attention not only in photocatalysis but also in optical sensors such as electrochemiluminescence (ECL) sensing of metal ions. However, when the concentrations of interfering metal ions are several times higher than that of the target metal ion, it is almost impossible to distinguish which metal ion changes the ECL signals in real sample detection. Herein, we report that the dual-ECL signals could be actuated by different ECL reactions merely from GPPCN nanosheets at anodic and cathodic potentials, respectively. Interestingly, the different metal ions exhibited distinct quenching/enhancement of the ECL signal at different driven potentials, presumably ascribed to the diversity of energy-level matches between the metal ions and GPPCN nanosheets and catalytic interactions of the intermediate species in ECL reactions. On this basis, without any labeling and masking reagents, the accuracy and reliability of sensors based on the ECL of GPPCN nanosheets toward metal ions were largely improved; thus, the false-positive result caused by interferential metal ions could be effectively avoided. As an example, the proposed GPPCN ECL sensor with a detection limit of 1.13 nM was successfully applied for the detection of trace Ni²⁺ ion in tap and lake water

Highly Selective and Sensitive Electrochemical Immunoassay of Cry1C Using Nanobody and π–π Stacked Graphene Oxide/Thionine Assembly

Cry1C is one of the emerging toxin proteins produced by the <i>Bacillus thuringiensis</i> in the genetically modified crops for pest control in agriculture; thus, it is vital to measure the Cry1C level in crops for the healthy and environmental concerns. Current detections of Cry1C mainly rely on instrumental analysis such as high-performance liquid chromatography, which are time-consuming and are generally cost-prohibitive. Herein, a simple nanobodies (Nbs)-based electrochemical immunosensor has been first proposed for highly selective and sensitive detection of Cry1C. The Nbs pair, i.e., Nb51 and Nb54, which bind to different epitopes on Cry1C, was screened out from an immunized Bactrian camel, with an extra benefit of higher stability compared with conventional antibodies. Further, by using a π–π stacked graphene oxide/thionine assembly that had fast electron transfer kinetics as an electroactive label, the immunoreaction that occurred between the two Nbs and Cry1C can be highly sensitively quantified by square wave voltammetry. The linear detection range was from 0.01 to 100 ng·mL^–1, and the low detection limit was 3.2 pg·mL^–1. This method was further successfully applied for sensing Cry 1C in spiked samples with recoveries ranging from 100.17% to 106.69% and relative standard deviation less than 4.62%. This proposed assay would provide a simple highly sensitive and selective approach for the Cry1C toxin detection and be applicable to be extended to other toxin proteins sensing in foods

Quantitative Evaluation of Biological Reaction Kinetics in Confined Nanospaces

Evaluating the kinetics of biological reaction occurring in confined nanospaces is of great significance in studying the molecular biological processes in vivo. Herein, we developed a nanochannel-based electrochemical reactor and a kinetic model to investigate the immunological reaction in confined nanochannels simply by the electrochemical method. As a result, except for the reaction kinetic constant that was previously studied, more insightful kinetic information such as the moving speed of the antibody and the immunological reaction progress in nanochannels were successfully revealed in a quantitative way for the first time. This study would not only pave the investigation of molecular biological processes in confined nanospaces but also be promising to extend to other fields such as biological detection and clinical diagnosis