Simultaneous Detection of Biomarkers in Urine Using a Multicalibration Potentiometric Sensing Array Combined with a Portable Analyzer.

Domestic monitoring devices make real-time and long-term health monitoring possible, allowing people to track their health status regularly. Uric acid (UA), creatinine, and urea in urine are three important biomarkers for various diseases, especially kidney diseases. This work proposed a 10-channel potentiometric sensing array containing a UA electrode group, a creatinine electrode group, a urea electrode group, a pH electrode group, and one pair of reference channels, which could be connected with a portable potentiometric analyzer, realizing the simultaneous detection of UA, creatinine, urea, and pH in urine. The prepared Pt/carbon nanotubes (CNTs)-uricase, creatinine deiminase, Au@urease, and polyaniline were employed as the sensing materials, showing responses to four targets with high sensitivity and selectivity. To improve the accuracy of domestic monitoring, a calibration channel was integrated into each electrode group to calibrate the basic potential of the sensing channels, and the influences of pH and temperature on the responses were investigated through the pH electrode group and an external temperature probe to calibrate the slope and intercept. With the preset of the deduced calibration parameters and computational formula for the four targets in the analyzer in Lab Mode, the concentrations of UA, creatinine, and urea and the pH of the human urine samples were directly displayed on the screen of the analyzer in Practical Mode. The agreement of these results with those obtained from commercial kits and pH meters reveals the high potential of these methods for developing domestic devices to facilitate health monitoring.

In Situ Investigation of Intercellular Signal Transduction Based on Detection of Extracellular pH and ROS by Scanning Electrochemical Microscopy.

Intercellular signal transduction plays an important role in the regulation of biological activities. Herein, a Transwell chamber-based two-layer device combined with scanning electrochemical microscopy (SECM) technology has been proposed for in situ investigation of intercellular signal transduction. The cells in the device were cultured on two layers: the lower layer was for signaling cells, and the upper layer was for signal-receiving cells. The extracellular pH (pHe) and ROS (reactive oxygen species, ROSe) were in situ monitored by SECM potentiometric mode and SECM-MPSW (multipotential step waveform), respectively. When the signaling cells, including MCF-7, HeLa, and HFF cells, were electrically stimulated, the ROS release of the signal-receiving cells was promoted. By detecting the pH at the cell surface, it was found that more H+ generated by the signaling cells and two cell layers at a shorter distance could both cause the signal-receiving cells to release more ROS, revealing that H+ is one of the signaling molecules of intercellular communication. This SECM-based in situ monitoring strategy provides an effective way to investigate intercellular signal transduction and explore the corresponding mechanism.

A Multicalibration Urea Potentiometric Sensing Array Based on Au@urease Nanoparticles and Its Application in Home Detection.

Home potentiometric sensing devices can real-time monitor personal health status and are widely used in the prevention and management of related diseases. However, variations in the composition and the pH of the sample matrix tend to change the basic potential and response slope of some potentiometric sensors, thus affecting detection reliability. Therefore, this work uses the detection of urea in urine as a model to improve reliability of the potentiometric sensor in home detection. Au@urease nanoparticles were synthesized as the sensing material to improve the stability of the urease-based potentiometric sensor. Meanwhile, a multicalibrated urea potential (MCUP) sensing array was designed, which consists of a urea electrode group, a pH electrode group, and a reference channel. The urea electrode group and the pH electrode group contain respectively a sensing channel and a calibration channel. The basic potential of sensing channels can be calibrated through the corresponding calibration channels. Moreover, the pH electrode group can not only measure the pH values of the samples but also calibrate the response slope of the urea electrode group through the calibration coefficient, thus improving the reliability of home detection. Consequently, the potentiometric sensing array based on the enzyme reaction can be applied in body fluids with a wide pH range.

In Situ Monitoring of Extracellular K+ Using the Potentiometric Mode of Scanning Electrochemical Microscopy with a Carbon-Based Potassium Ion-Selective Tip.

The expression of potassium channels can be related to the occurrence and development of tumors. Their change would affect K+ outflow. Thus, in situ monitoring of extracellular K+ shows a great significance. Herein, the dual-functional K+ ion-selective electrode as the scanning electrochemical microscopy (SECM) tip (K+-ISE SECM tip) has been developed for in situ monitoring of the extracellular K+. Based on multi-wall carbon nanotubes as a transduction layer, the K+-ISE SECM tip realizes both the plotting of approach curves to position the tip for in situ detection and the recording of potential responses. It shows a near Nernstian response, good selectivity, and excellent stability. Based on these characteristics, it was used to in situ monitor K+ concentrations ([K+]o) of three breast cancer cell lines (MCF-7, MDA-MB-231, and SK-BR-3 cells) at 3 µm above the cell, and [K+]o of MDA-MB-231 cells show the highest value, followed by MCF-7 cells and SK-BR-3 cells. K+ outflow induced by electrical stimulation or pH changes of the culture environment (Δ[K+]o) was further determined, and the possible mechanism of K+ outflow was investigated with 4-aminopyridin (4-AP). MCF-7 cells present the largest value of Δ[K+]o, followed by MDA-MB-231 cells and SK-BR-3 cells at all the stimulation potentials, and pH 6.50 shows the greatest impact on K+ outflow of the three cell lines. The pretreatment of 4-AP changed K+ outflow, probably due to the regulation of voltage-gated channels. These findings provide insight into a deep understanding of the microenvironment influence on K+ outflow, thereby reflecting the possible mechanism of potassium channels.

Home Detection Technique for Na+ and K+ in Urine Using a Self-Calibrated all-Solid-State Ion-Selective Electrode Array Based on Polystyrene-Au Ion-Sensing Nanocomposites.

An all-solid-state ion-selective electrode (ASS-ISE) array that is portable and easily miniaturized can meet the needs of home sensing devices for long-term health monitoring. However, their stability and accuracy are affected by the multistep modification required for ASS-ISE manufacturing and the complex background signal of real samples. In this study, a four-channel ISE array with the integration of a calibration channel has been developed based on polystyrene-Au (PS-Au) ion-sensing nanocomposites (PS-Au ISE array) for the home detection of Na+ and K+. The nanocomposites combine target recognition function and ion-electron transduction function and could be modified on the channel surface by direct drop-casting, thus simplifying the preparation process and then improving the stability. Meanwhile, the integrated calibration channel could automatically deduct complex background signals in real sample analysis and thus improve the accuracy. As a result, the proposed self-calibrated PS-Au ISE array showed a near Nernstian behavior for Na+ and K+ in the range of 1 × 10-2 M-1 × 10-4 M, and the detection limits were 6.8 × 10-5 M and 5.5 × 10-5 M in artificial urine. The linear equations can be obtained according to the slopes and intercepts of Na+ and K+, and thus, the concentration of the target ions can be directly read out by combining this PS-Au ISE array with the smart electronic device. Furthermore, the detection results of Na+ and K+ in human urine agreed well with those obtained by ICP-AES, suggesting that this proposed self-calibrated PS-Au ISE array is very suitable for home smart sensing devices, facilitating the health monitoring.

Study on Defective T Junction-Mediated Strand Displacement Amplification and Its Application in Microchip Electrophoretic Detection of Longer Bacterial 16S rDNA.

Current strand displacement amplification (SDA)-based nucleic acid sensing methods generally rely on a ssDNA template that involves complementary bases to the endonuclease recognition sequence, which has the limitation of detecting only short nucleic acids. Herein, a new SDA method in which the defective T junction structure is first used to support SDA (dT-SDA) was proposed and applied in longer DNA detection. In dT-SDA, an auxiliary probe and a primer were designed to specifically identify the target gene, following the formation of a stable defective T junction structure through proximity hybridization, and the formation of defective T junctions could further trigger cascade SDA cycling to produce numerous ssDNA products. The quantity of these ssDNA products was detected through microchip electrophoresis (MCE) and could be transformed to the concentration of the target gene. Moreover, the applicability of this developed strategy in detecting long genomic DNA was verified by detecting bacterial 16S rDNA. This proposed dT-SDA strategy consumes less time and has satisfactory sensitivity, which has great potential for effective bacterial screening and infection diagnosis.

In situ monitoring of the effect of Cu2+ on the membrane permeability of a single living cell with a dual-electrode tip of a scanning electrochemical microscope.

Here, an Au-Cu dual-electrode tip was designed to monitor the effect of Cu2+ on the membrane permeability of a single living cell in situ using scanning electrochemical microscopy. The probe approach curves (PACs) were obtained using potassium ferricyanide as a redox mediator. Meanwhile, according to the simulation, theoretical PACs could be acquired. Thus, the cell membrane permeability coefficient (Pm) values were obtained by overlapping the experimental PACs with the theoretical values. Cu2+ was directly generated by electrolyzing the Cu electrode of the dual-electrode tip to investigate its effect on the cell membrane permeability in situ. This work has potential value to improve the understanding of the mechanism of acute heavy metal damage on the cell membrane and will also help clarify the role of heavy metal ions in physiological or pathological processes.

Simultaneous electrochemical determination of nuc and mecA genes for identification of methicillin-resistant Staphylococcus aureus using N-doped porous carbon and DNA-modified MOF.

The detection of Staphylococcus aureus specific gene in combination with the mecA gene is vitally important for accurate identification of methicillin-resistant Staphylococcus aureus (MRSA). A homogeneous electrochemical DNA sensor was fabricated for simultaneous detection of mecA and nuc gene in MRSA. Metal-organic framework (type UiO-66-NH2) was applied as nanocarrier. Two electroactive dyes, methylene blue (MB) and epirubicin (EP), were encapsulated in UiO-66-NH2, respectively, and were locked by the hybrid double-stranded DNA. Based on the target-response electroactive dye release strategy, once target DNA exists, it completely hybridizes with displacement DNA (DEP and DMB). So DEP and DMB is displaced from the MOF surface, causing the release of electroactive dyes. Co-Zn bimetallic zeolitic imidazolate framework-derived N-doped porous carbon serves for electrode modification to improve electrocatalytic performance and sensitivity. The differential pulse voltammetry peak currents of MB and EP were accurately detected at - 0.14 V and - 0.53 V versus the Ag/AgCl reference electrode, respectively. Under the optimal conditions, the detection limits of mecA gene and nuc gene were 3.7 fM and 1.6 fM, respectively. Combining the effective application of MOFs and the homogeneous detection strategy, the sensor exhibited satisfactory performance for MRSA identification in real samples. The recovery was 92.6-103%, and the relative standard deviation was less than 5%. Besides, MRSA and SA can also be distinguished. This sensor has great potential in practical applications.

Magneto-Mediated Electrochemical Sensor for Simultaneous Analysis of Breast Cancer Exosomal Proteins.

Breast cancer is a heterogeneous disease, and it lacks special tumor markers. Exosomes, new noninvasive biomarkers, with the proteins on the exosome surface show potential for the diagnosis and prognosis of a tumor. However, assessing the variations of exosomal proteins still faces significant challenges. Herein, a magneto-mediated electrochemical sensor based on host-guest recognition has been developed for simultaneous analysis of breast cancer exosomal proteins. Magnetic beads (MB) modified with CD63 aptamer was first employed to capture exosomes. Silica nanoparticles (SiO2 NPs) was modified with MUC1, HER2, EpCAM, and CEA aptamers for specific exosomal proteins identification, respectively, and functionalized with N-(2-((2-aminoethyl)disulfanyl)ethyl) ferrocene carboxamide (FcNHSSNH2) as the signal molecule. The sandwich structure (MB-exosomes-SiO2 NPs probe) was separated by a magnet, and N-(2-mercaptoethyl) ferrocene carboxamide (FcNHSH) was released to the supernatant by the addition of reductants (dithiothreitol, DTT) that break the disulfide bond of FcNHSSNH2. FcNHSH and the graphene oxide-cucurbit [7](GO-CB[7]) modified screen-printed carbon electrode (SPCE) was employed to monitor the oxidation current signals. In this way, four tumor markers on different breast cancer cells (MCF-7, SK-BR-3, MDA-MB-231, and BT474) derived exosomes were sensitively detected. Furthermore, the present assay enabled accurate analysis of exosomes from breast cancer patients, suggesting the potential of exosome analysis in clinic diagnosis.

Identification of Cell Status via Simultaneous Multitarget Imaging Using Programmable Scanning Electrochemical Microscopy.

A programmable multitarget-response electrochemical imaging technique was presented using scanning electrochemical microscopy (SECM) combined with a self-designed waveform. The potential waveform applied to the tip decreased the charging current caused by the potential switch, enhancing the signal-to-noise ratio. This programmable SECM (P-SECM) method was used to scan a metal strip for verifying its feasibility in feedback mode. Since it could achieve simultaneous multitarget imaging during one single imaging process, PC12 cells status was imaged and identified through three different molecules (FcMeOH, Ru(NH3)63+, and oxygen). The FcMeOH image eliminated the error from cell height, and the Ru(NH3)63+ image verified the change of membrane permeability. Moreover, the oxygen image demonstrated the bioactivity of the cell via its intensity of respiration. Combining information from these three molecules, the cell status could be determined accurately and also the error caused by time consumption with multiple scans in traditional SECM was eliminated.

Real-time monitoring of extracellular pH using a pH-potentiometric sensing SECM dual-microelectrode.

Extracellular pH can indicate the variation in organelle function and cell state. It is important to measure extracellular pH (pHe) with a controllable distance. In this work, a potentiometric SECM dual-microelectrode was developed to monitor the pHe of MCF-7 cells under electrical stimulation. The distance between the dual-microelectrode and the cells was determined first with a gold microelectrode by recording the approaching curve, and the pH was determined using an open-circuit potential (OCP) technique with a polyaniline-modified Pt microelectrode. The pH microelectrode showed a response slope of 53.0 ± 0.4 mV/pH and good reversibility from pH 4 to pH 8, fast response within 10 s, and a potential drift of 1.13% for 3 h, and thus was employed to monitor the pHe of stimulated cells. The value of pHe decreased with the decrease in the distance to cells, likely due to the release of H+. With an increase in the stimulation potential or time, the pHe value decreased, as the cell membrane became more permeable, which was verified by fluorescence staining of calcein-AM/PI (propidium iodide). Based on these results, this method can be widely applied for determining the species released by biosystems at a controllable position.

An electrochemical sensor for bacterial lipopolysaccharide detection based on dual functional Cu2+-modified metal-organic framework nanoparticles.

An electrochemical sensor based on dual functional Cu2+-modified metal-organic framework nanoparticles (Cu2+-NMOFs) for sensitive detection of bacterial lipopolysaccharide (LPS) is reported. Cu2+-NMOFs were prepared and characterized by SEM, EDS, XRD, and XPS. In this LPS sensor, LPS firstly immobilized in gold nanoparticles/reduced graphene oxide by C18 alkane thiol chains, since the LPS can interact with the C18 alkyl chains by strong intermolecular interactions. Then the Cu2+-NMOFs were captured by the anionic groups of the carbohydrate portions of LPS molecules and played a vital role of recognition unit. More importantly, the Cu2+-NMOFs can catalyze dopamine oxidation to generate aminochrome, resulting in a strong electrochemical oxidation signal. The electrochemical sensor based on dual functional Cu2+-NMOFs was investigated by differential pulse voltammetry, and the stripping peak currents of dopamine oxidized to aminochrome were used to monitor the level of LPS. The developed method demonstrated a wide linear range from 0.0015 to 750 ng/mL with a limit of detection of 6.1 × 10-4 ng/mL. The fabricated sensor was applied to detect LPS in mouse blood serum and satisfactory results were achieved. Compared to other detection schemes by using the LPS-binding proteins, peptides, and aptamer, the proposed LPS determination based on the catalytic peroxidase-mimicking NMOFs has some advantages such as good reproducibility, low detection limit, and excellent specificity. Graphical abstract An electrochemical sensor based on dual functional Cu2+-modified metal-organic framework was developed for detection of bacterial lipopolysaccharide. This sensor combined a metal ion-based target recognition and electrocatalytic detection, and provided a high sensitive strategy for detection of lipopolysaccharide.

A microchip electrophoretic assay for DNA methyltransferase activity based on methylation-sensitive endonuclease Dpnâ ¡.

DNA methylation is a significant epigenetic modification and the methods for the detection of DNA methyltransferase (MTase) activity are important due to aberrant methylation closely related to the occurrence of cancer. In this study, a simple and rapid microchip electrophoresis (ME) coupled with LED-induced fluorescence (LEDIF) method was presented for the detection of Dam MTase activity. This strategy was based on methylation-sensitive endonuclease DpnⅡ which could recognize the same specific site 5'-GATC-3' with Dam MTase in double-stranded DNA (dsDNA). The adenines in the specific site could be methylated by Dam MTase, then the special site could not be digested by DpnⅡ. Both methylated dsDNA and unmethylated dsDNA could be analyzed by ME-LEDIF after stained by SYBR gold. The results showed the fluorescence intensities of methylated dsDNA were directly proportional to Dam MTase activities in the range of 0.5-20 U/mL with a detection limit of 0.12 U/mL. Furthermore, the method could successfully be applied to evaluation experiments of Dam MTase inhibitors. The results confirmed the ME-LEDIF method is a promising approach for inhibitors screening of DNA MTase and development of anticancer drugs.

Selective detection of cytochrome C by microchip electrophoresis based on an aptamer strategy.

The release of cytochrome C (Cyt C) plays an important role in apoptosis. In this study, selective and sensitive detection of Cyt C based on an aptamer strategy coupled with MCE was developed. Following the binding of a specific aptamer to Cyt C, the aptamer exhibited an irregular state, reducing the binding affinity of a fluorescent probe, and thus preventing the aptamer-Cyt C complexes from detection within the MCE. The height of the detection peak of the residual aptamer linearly decreased, and therefore, the difference in peak height of residual aptamer compared to that of the initial aptamer was used to quantify the captured protein concentration. Experimental conditions such as incubation time, pH, temperature, and ionic strength were optimized. A measurement of Cyt C concentration by MCE was achieved within 135 s, with a limit of detection as low as 0.4 nM. The proposed method has high selectivity and good stability for the detection of Cyt C. The experimental results demonstrate that this method is quick, consumes only a small quantity of sample, is highly selectivity and exhibits high sensitivity.

Hierarchical Assembly of Peptoid-Based Cylindrical Micelles Exhibiting Efficient Resonance Energy Transfer in Aqueous Solution.

Herein we show that by appending bulky ß-cyclodextrin (CD) groups onto sheet-forming peptoids, we obtain cylindrical micelles that further assembly into membranes and intertwined ribbons on substrates in aqueous solution, depending on the choice of solution and substrate conditions. In situ atomic force microscopy (AFM) shows that micelle assembly occurs in two steps, starting with "precursor" particles that transform into worm-like micelles, which extend and coalesce to form the higher order structures with a rate and a degree of cooperativity dependent on pH and Ca2+ concentration. After co-assembly with hydrophobic 4-(2-hydroxyethylamino)-7-nitro-2,1,3-benzoxadiazole (NBD) donors that occupy the hydrophobic core, followed by exposure to hydrophilic Rhodamine B as acceptors that insert into cyclodextrin, the micelles exhibit highly efficient Förster resonance energy transfer efficiency in aqueous solution, thereby mimicking natural light harvesting systems.

Internal Calibration Potentiometric Aptasensors for Simultaneous Detection of Hg2+, Cd2+, and As3+ Based on a Screen-Printed Carbon Electrodes Array.

An all-solid-state potentiometric aptasensor array based on a multichannel disposable screen-printed carbon electrode (SPCE) was demonstrated for the simultaneous detection of Hg2+, Cd2+, and As3+ by open circuit potential (OCP) technology. The potential of the channel with an internal calibration DNA sequence (IC-DNA) was employed as the internal calibration potential (ICP) to subtract the background signal generated by the detection system, providing a built-in correction methodology. As a result, the developed aptasensor array showed high sensitivity and accuracy for detecting Hg2+, Cd2+, and As3+ without mutual interference or interference from other ions. The linear response ranged from 2.5 pM to 2.5 µM, and the detection limits for Hg2+, Cd2+, and As3+ were 2.0, 0.62, and 0.17 pM, respectively. Furthermore, the potentiometric aptasensor array was successfully applied for the simultaneous detection of three ions in real samples. The results obtained from the developed approach agreed well with the results obtained from inductively coupled plasma mass spectrometry.

A homochiral porous organic cage with large cavity and pore windows for the efficient gas chromatography separation of enantiomers and positional isomers.

Porous organic cages composed of discrete cage molecules have attracted considerable recent attention as gas adsorption materials and separation media. In this study, we report a homochiral porous organic cage CC5 with a large cavity and pore windows as a novel stationary phase for high-resolution gas chromatographic separations. The capillary column was prepared by a static coating method. A large number of racemic compounds have been resolved on the coated capillary column, including derivatized amino acids, alcohols, alcohol amines, esters, ethers, ketones, and epoxides. It is interesting that the CC5-coated capillary column exhibits significant chiral recognition complementarity to a commercial ß-DEX 120 column and a previously reported homochiral porous organic cage CC3-R-coated column, which could expand the range of the analytes amenable to separation on porous organic cage-based capillary columns. Moreover, the fabricated column also shows excellent selectivity for the separation of positional isomers, including the challenging ethylbenzene and xylene isomers. Experimental results demonstrate an excellent separation performance and stability of the CC5-coated column, making it promising for gas chromatography applications.

Synthesis and characterization of a multimode stationary phase: Congo red derivatized silica in nano-flow HPLC.

A novel Congo red (CR) derivatized silica stationary phase was prepared and packed into a fused silica capillary tube for nano-flow HPLC. A variety of analytes including poly-aromatic hydrocarbons, parabens, acids, sulfonamides, bases, and nucleosides were successfully separated using the CR. In comparison with commercial ODS columns, this new stationary phase has a different separation mechanism (hydrophobically-assisted ion-exchange), which was evident in the separation of benzoic acid derivatives and sulfonamides. The successful application of CR-bonded silica stationary phase in the HILIC and PALC modes demonstrates the effectiveness of this potential chromatographic material in nano flow HPLC.

Accurate data process for nanopore analysis.

Data analysis for nanopore experiments remains a fundamental and technological challenge because of the large data volume, the presence of unavoidable noise, and the filtering effect. Here, we present an accurate and robust data process that recognizes the current blockades and enables evaluation of the dwell time and current amplitude through a novel second-order-differential-based calibration method and an integration method, respectively. We applied the developed data process to analyze both generated blockages and experimental data. Compared to the results obtained using the conventional method, those obtained using the new method provided a significant increase in the accuracy of nanopore measurements.

Homochiral Porous Organic Cage with High Selectivity for the Separation of Racemates in Gas Chromatography.

Porous organic molecular cages as a new type of porous materials have attracted a tremendous attention for their potential applications in recent years. Here we report the use of a homochiral porous organic cage (POC) (CC3-R) diluted with a polysiloxane (OV-1701) as a stationary phase for high-resolution gas chromatography (GC) with excellent enantioselectivity. A large number of optical isomers have been resolved without derivatization, including chiral alcohols, diols, amines, alcohol amines, esters, ketones, ethers, halohydrocarbons, organic acids, amino acid methyl esters, and sulfoxides. Compared with commercial ß-DEX 120 and Chirasil-L-Val columns, the CC3-R coated capillary column offered more preeminent enantioselectivity. In addition, CC3-R also exhibits good selectivity for the separation of isomers, linear alkanes, alcohols, and aromatic hydrocarbons. The excellent resolution ability, repeatability, and thermal stability make CC3-R a promising candidate as a novel stationary phase for GC. The study described herein first proves useful commercially. This work also indicates that porous organic molecular materials will become more attractive in separation science.