Quantification of multiple microRNAs by microchip electrophoresis assisted by strand displacement amplification.

MicroRNAs (miRNAs) are increasingly recognized as potential biomarkers for the early diagnosis of cancer. However, the concurrent detection of multiple miRNAs in biological samples presents a significant challenge due to their high homogeneity and low abundance. This study introduced a novel approach combining strand displacement amplification (SDA) with microchip electrophoresis (MCE) for the simultaneous quantitation of trace levels of three miRNAs associated with cancer: miRNA-21, miRNA-145, and miRNA-221. Specifically designed probes were utilized to selectively capture the target miRNAs, thereby initiating the SDA process in a single solution without cross-interference. Under optimized conditions, the SDA-MCE method achieved the limit of detection (LOD) as low as 0.02 fM (S/N = 3) and the limit of quantitation (LOQ) as low as 0.1 fM across a broad linear range spanning from 0.1 fM to 1 pM. The SDA reaction was completed in approximately 1.5 h, and all target products were separated within 135 s through MCE. Application of this method for the simultaneous detection of these three miRNAs in human lung cancer cell samples yielded satisfactory results. Featuring high sensitivity, rapid analysis, minimal reagent consumption, and straightforward operation, the proposed MCE-SDA strategy holds considerable promise for multi-miRNAs detection applications.

Isothermal strand displacement polymerase reaction (ISDPR)-assisted microchip electrophoresis for highly sensitive detection of cancer associated microRNAs.

More and more studies have found that microRNAs (miRNAs) are markers of cancer, and detection of miRNAs is beneficial for early diagnosis and prognosis of cancer. In this paper, the isothermal strand displacement polymerase reaction (ISDPR), which is an enzyme-assisted nucleic acid amplification method, was studied to combine with microchip electrophoresis (MCE) for a simultaneously detection of two cancer related miRNAs named microRNA-21 (miR-21) and microRNA-221 (miR-221). In the ISDPR amplification, two different DNA hairpins (HPs) were specifically designed, so that miR-21 and miR-221 could respectively bind to HPs and started ISDPR amplification to generate two different products which were ultimately detected by MCE. The optimal conditions of ISDPR were carefully investigated, and the limits of detection (LOD) of miR-21 and miR-221 were as low as 0.35 fM and 0.25 fM (S/N = 3) respectively under these conditions. The human lung tumor cells and serum samples were analyzed by this ISDPR-MCE method and satisfactory results were obtained, which means that this method is of high sensitivity, high efficiency, low reagent consumption and simple operation in miRNAs detection.

Aptamer-mediated double strand displacement amplification with microchip electrophoresis for ultrasensitive detection of Salmonella typhimurium.

Rapid and quantitative detection of foodborne bacteria is of great significance to public health. In this work, an aptamer-mediated double strand displacement amplification (SDA) strategy was first explored to couple with microchip electrophoresis (MCE) for rapid and ultrasensitive detection of Salmonella typhimurium (S. Typhimurium). In double-SDA, a bacteria-identified probe consisting of the aptamer (Apt) and trigger sequence (Tr) was ingeniously designed. The aptamer showed high affinity to the S. Typhimurium, releasing the Tr sequence from the probe. The released Tr hybridized with template C1 chain, initiating the first SDA to produce numerous output strands (OS). The second SDA process was induced with the hybridization of the liberated OS and template C2 sequence, generating a large number of reporter strands (RS), which were separated and quantified through MCE. Cascade signal amplification and rapid separation of nucleic acids could be realized by the proposed double-SDA method with MCE, achieving the limit of detection for S. typhimurium down to 6 CFU/mL under the optimal conditions. Based on the elaborate design of the probes, the double-SDA assisted MCE strategy achieved better amplification performance, showing high separation efficiency and simple operation, which has satisfactory expectation for bacterial disease diagnosis.

Cyclic Multiple Primer Generation Rolling Circle Amplification Assisted Capillary Electrophoresis for Simultaneous and Ultrasensitive Detection of Multiple Pathogenic Bacteria.

Efficient, accurate, and economical detection of pathogenic bacteria is crucial in ensuring food safety and preventing foodborne illnesses. How to fulfill the highly sensitive and simultaneous detection of multiple trace pathogenic bacteria is a big challenge. In this work, capillary electrophoresis coupled with a cyclic multiple primer generation rolling circle amplification (cyclic MPG-RCA) was studied for highly sensitive and simultaneous detection of three kinds of pathogenic bacteria. The cyclic MPG-RCA was based on a carefully designed clover-shaped DNA probe, in which three "leaves" corresponded to three types of aimed pathogenic bacteria: Shigella dysenteriae (S. dysenteriae), Salmonella enterica subsp. enterica serovar Typhi (S. Typhi), and Vibrio parahaemolyticus (V. parahaemolyticus). Under the optimal experimental conditions, the limits of detection (S/N = 3) of this method for bacterial target DNA were 11.4 amol·L-1 (S. dysenteriae), 4.88 amol·L-1 (S. Typhi), and 14.9 amol·L-1 (V. parahaemolyticus), and the conversion concentrations for the target bacteria were 10 colony-forming units (CFU)·mL-1 (S. dysenteriae), 3 CFU·mL-1 (S. Typhi), and 12 CFU·mL-1 (V. parahaemolyticus). This method had been applied to the detection of tap water samples with good results, which proved that it could be used as an effective tool for trace pathogenic bacteria monitoring in foods, environments, and medicines.

Detection of Escherichia coli by capillary electrophoresis assisted by large volume sample stacking and nicking endonuclease signal amplification.

Efficient, accurate and economical detection of pathogenic bacteria is crucial in ensuring food safety and preventing foodborne illnesses. In this study, a capillary electrophoresis coupled laser-induced fluorescence assay (CE-LIF) was developed for the detection of Escherichia coli (E. coli) by detecting its specific DNA. The CE-LIF was assisted by both online enrichment and offline amplification to improve the detection sensitivity of bacterial DNA. Here the online amplification was performed by large volume sample stacking (LVSS), while the offline amplification was nicking endonuclease signal amplification (NESA). Under the optimal experimental conditions, the detection limit of bacterial target DNA was 2.5 fM, and the conversion concentration of E. coli was 3 CFU · mL-1. The method had been applied to the detection of commercially available skim milk samples with good results, which proved that it could be used as an effective tool for food and environmental bacteria monitoring.

Propensity-matched analysis of robotic versus sternotomy approaches for mitral valve replacement.

To compare early and medium-term outcomes between robotic and sternotomy approaches for mitral valve replacement (MVR). Clinical data of 1393 cases who underwent MVR between January 2014 and January 2023 were collected and stratified into robotic MVR (n = 186) and conventional sternotomy MVR (n = 1207) groups. The baseline data of the two groups of patients were corrected by the propensity score matching (PSM) method. After matching, the baseline characteristics were not significant different between the two groups (standardized mean difference < 10%). Moreover, the rates of operative mortality (P = 0.663), permanent stroke (P = 0.914), renal failure (P = 0.758), pneumonia (P = 0.722), and reoperation (P = 0.509) were not significantly different. Operation, CPB and cross-clamp time were shorter in the sternotomy group. On the other hand, ICU stay time, post-operative LOS, intraoperative transfusion, and intraoperative blood loss were shorter or less in the robot group. Operation, CPB, and cross-clamp time in robot group were all remarkably improved with experience. Finally, all-cause mortality (P = 0.633), redo mitral valve surgery (P = 0.739), and valve-related complications (P = 0.866) in 5 years of follow-up were not different between the two groups. Robotic MVR is safe, feasible, and reproducible for carefully selected patients with good operative outcomes and medium-term clinical outcomes.

Ultrasensitive detection of exosomes by microchip electrophoresis combining with triple amplification strategies.

The analysis of exosomes is significant as they can be used for various pathophysiological processes, especially cancer related intercellular communication. Therefore, a convenient, reliable, and sensitive detection method is urgently needed. Strand displacement amplification (SDA) and catalytic hairpin assembly (CHA) are two kinds of effective isothermal nucleic acid amplification methods. In this article, an efficient quantitative MCE method for detecting human breast cancer cell (MCF-7) exosomes assisted by triple amplification strategies combining cholesterol probe (Chol-probe) with SDA-CHA was first developed. CD63 aptamer was immobilized on the avidin magnetic beads to specifically capture exosomes and then Chol-probe with high affinity was spontaneously inserted into the exosome membrane, which was the first step of amplification strategy to improve detection sensitivity. After magnetic separation, Chol-probe could complement ssDNA and trigger SDA, producing a large number of DNA sequences (Ta) to trigger CHA, achieving SDA-CHA amplification. Under optimal conditions, the detection limit (LOD) for MCF-7 exosomes was as low as 26 particle/µL (S/N = 3). This method provides an effective approach for sensitive and accurate quantification of tumor exosomes, and can be expected to detect exosomes in clinical samples.

Effects of different unconventional feed combinations on growth, digestion and rumen fermentation of dairy cows.

To explore the effects of unconventional feed combinations on the growth and production, digestion and metabolism, and rumen fermentation of dairy cows, three different dairy cows were selected for the experiment. They are Holstein cows with permanent rumen fistula, 3 primiparous cows and 6 multiparous cows. The cow diet was prepared according to the ratio of 0% CGF, 7% CGF and 11% CGF. Part of alfalfa hay in the conventional diet was replaced by CGF and Leymus chinensis. The study analyzed the feed intake, digestibility, lactation performance, blood biochemical indicators, rumen degradation parameters, rumen microorganisms and other indicators of dairy cows. The nutritional composition, digestible nutrients and absorbable protein content of CGF, L. chinensis and alfalfa hay were verified. The feed economic benefits of different unconventional feed combinations were also investigated. The protein small intestine digestibility of CGF was higher than that of alfalfa hay. tdFA, NEm, NEg, and DEp were significantly higher than those of L. chinensis and alfalfa hay (P<0.05). Under the three CGF ratios, the nutrient intake and digestibility of the CGF-11% group were the highest (P<0.05). The S, Kd dry matter degradation rate and crude protein degradation rate of the CGF-11% group were significantly higher than those of the CGF-0% group and CGF-7% group (P<0.05). The CGF-11% group had the highest total output value and economic benefits, 119.057 ¥/d and 68.62 ¥/d respectively. To sum up, it was feasible to use the combination of CGF and L. chinensis to replace part of alfalfa hay in cow feed. This method could effectively promote rumen degradation and nutrient absorption in dairy cows. And it can improve the production and economic benefits of dairy farming. This is of great value for adjusting the structure of aquaculture feed in China.

A multi-calibration potentiometric sensing array based on diboronic acid-PtAu/CNTs nanozyme for home monitoring of urine glucose.

All-solid-state potentiometric sensors that are easily miniaturized and arrayed are widely used in home diagnostics. However, changes in sample matrix compositions tend to affect the basic potential of the potentiometric sensor, and pH of sample could change the response slope, thus affecting the detection reliability. This study takes the detection of glucose in urine as a model to increase the reliability of potentiometric sensors in home detection. PtAu/CNTs nanozyme modified by diboronic acid has been designed, showing better catalytic selectivity for glucose by experiments and theoretical calculations. Moreover, glucose electrode group in a multi-calibration glucose potentiometric sensing array can realize the basic potential calibration of sensing channel by the calibration channel. Meanwhile, the pH electrode group can not only measure the urine pH, but also calibrate the response slope of the glucose electrode group, thus improving the reliability of home detection.

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.

Highly sensitive detection of MUC1 by microchip electrophoresis combining with target recycling amplification and strand displacement amplification.

Mucin 1 (MUC1) is usually overexpressed in a variety of malignant tumors, and quantitative analysis of MUC1 plays an important role in the early diagnosis of cancer. In this work, a highly sensitive MUC1 assay was developed by integrating microchip electrophoresis (MCE) with target recycling amplification (TRA) and strand displacement amplification (SDA). Specifically, the presence of MUC1 can trigger the exposure of the designed hairpin probe (HP) to initiate SDA and an amplified amount of ssDNA is produced finally. The amount of these ssDNA can be detected by MCE, then the concentration of MUC1 can be obtained through the correlation between MUC1 concentration and ssDNA concentration. The experimental results show that the MCE signal had a good linear relationship with MUC1 concentration in the range of 1.0 pg/mL - 1.0 × 103 pg/mL with a low limit of detection of 0.23 pg/mL under the optimal conditions (S/N = 3). Additionally, the assay had been successfully applied to detect MUC1 in biological samples with satisfactory results, providing an alternative assay for the detection of other tumor markers owing to the high sensitivity, high selectivity, simple operation and low sample consumption.

Biosensing bacterial 16S rDNA by microchip electrophoresis combined with a CRISPR system based on real-time crRNA/Cas12a formation.

The accurate, simple and sensitive detection of bacterial infections at the early stage is highly valuable in preventing the spread of disease. Recently, CRISPR-Cas12a enzyme-derived nucleic acid detection methods have emerged along with the discovery of the indiscriminate single-stranded DNA (ssDNA) cleavage activity of Cas12a. These nucleic acid detection methods are made effective and sensitive by combining them with isothermal amplification technologies. However, most of the proposed CRISPR-Cas12a strategies involve Cas-crRNA complexes in the preassembled mode, which result in inevitable nonspecific background signals. Besides, the signal ssDNA used in these strategies needs tedious pre-labeling of the signal molecules. Herein, a post-assembly CRISPR-Cas12a method has been proposed based on target-induced transcription amplification and real-time crRNA generation for bacterial 16S rDNA biosensing. This strategy is label-free through the combination of microchip electrophoresis (MCE) detection. In addition, this method eliminates the need for a protospacer adjacent motif (PAM) on the target sequences, and has the potential to be an effective and simple method for nucleic acid detection and infectious disease diagnosis.

Construction of Pd Single Site Anchored on Nitrogen-Doped Porous Carbon and Its Application for Total Antioxidant Level Detection.

Natural enzymes have excellent catalytic activity. However, due to their unstable nature and high cost, current research has turned to the synthesis and development of enzyme-like nanomaterials and single-atomic nanozymes. In this study, a single-atomic palladium-loaded nitrogen-doped porous carbon catalyst (SA-Pd/NPC) was prepared and used as a mimetic peroxidase to catalyze the substrates oxidation. The catalytic capability of the SA-Pd/NPC was tested by the TMB-H2O2 system, and it expressed a superior catalytic capability owing to the plentiful catalytic centers of the single-atom Pd, its high porosity, the large specific surface area, and the strong electron transfer capability of the NPC. For the color reaction of TMB, thiol antioxidants (e.g., glutathione, GSH) and non-thiol antioxidants (e.g., ascorbic acid, AA) are suitable for different inhibition mechanisms. GSH and AA are typical substances of these two main antioxidant types, respectively. Here, we demonstrate that this prepared catalyst could be used to simultaneously determine a variety of major known physiologically relevant thiol-containing and thiol-free antioxidants, accompanied by a blue color gradient change with UV-Vis spectra at 652 nm through the SA-Pd/NPC-catalyzed TMB-H2O2 system. Linear responses to GSH and AA could be obtained in the concentration ranges of 0.01-0.10 mM and 1-13 µM (both R2 values were greater than 0.970), respectively, while the limits of detection were 3 µM and 0.3 µM, respectively. The ability of the nanozyme to detect overall antioxidant levels (TAL) was also confirmed in subsequent tests on artificial saliva and biological samples.

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.

A ratiometric electrochemical biosensor for glycated albumin detection based on enhanced nanozyme catalysis of cuprous oxide-modified reduced graphene oxide nanocomposites.

Nanozymes have enzyme-like characteristics and nanozyme-based electrochemical sensors have been widely studied for biomarker detection. In this work, cuprous oxide-modified reduced graphene oxide (Cu2O-rGO) nanozyme was prepared by simultaneous reduction of copper chloride and graphene oxide. This Cu2O-rGO nanozyme displayed an outstanding electrocatalytic activity to glucose oxidation and was used as the modified material of a glassy carbon electrode to fabricate an electrochemical ratiometric biosensor for glycated albumin (GA) detection. In this ratiometric biosensor, methylene blue-labeled DNA tripods (MB-tDNA) were adsorbed on the Cu2O-rGO/GCE surface to form a bioinspired electrode (MB-tDNA/Cu2O-rGO/GCE), in which the catalytic sites of Cu2O-rGO were covered by MB-tDNA. In the presence of target GA, GA could be identified by the aptamer sequence contained in MB-tDNA, and a MB-tDNA/GA complex was formed and released into the solution, so the reduced current of MB-tDNA was decreased. Simultaneously, the oxidized current of the outer added glucose was increased since more catalytic sites of Cu2O-rGO nanozyme on the substrate electrode surface were exposed. The ratio of the peak currents of glucose oxidation and methylene blue reduction (IGlu/IMB) was used to monitor the GA level and ultimately improve the accuracy of the method. The electrochemical sensor showed a low detection limit of 0.007 µg mL-1 and a wide linear range from 0.02 to 1500 µg mL-1. The proposed sensor was also successfully used to measure the GA expression level in the blood serum of a diabetic mouse model.

Review of microchip analytical methods for the determination of pathogenic Escherichia coli.

Bacterial infections remain the principal cause of mortality worldwide, making the detection of pathogenic bacteria highly important, especially Escherichia coli (E. coli). Current E. coli detection methods are labour-intensive, time-consuming, or require expensive instrumentation, making it critical to develop new strategies that are sensitive and specific. Microchips are an automated analytical technique used to analyse food based on their separation efficiency and low analyte consumption, which make them the preferred method to detect pathogenic bacteria. This review presents an overview of microchip-based analytical methods for analysing E. coli, which were published in recent years. Specifically, this review focuses on current research based on microchips for the detection of E. coli and reviews the limitations of microchip-based methods and future perspectives for the analysis of pathogenic bacteria.

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.

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.

Ultrasensitive microchip electrophoretic detection of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) based on isothermal strand-displacement polymerase reaction.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens involved in hospital and community infection. To rapidly and sensitively detect the mecA gene, which is relevant to methicillin-resistant strains, microchip electrophoresis (MCE) integrated with isothermal strand-displacement polymerase reaction (ISDPR) was developed. In the ISDPR signal recycle amplification, the target DNA opened the DNA hairpin structure by specifically binding with the hairpin probe (HP), and then the primer hybridized with the probe and released the target DNA in the presence of Klenow Fragment exo- (KF exo-) polymerase. The released target DNA hybridized with the next HP and then was displaced by the primer again, consequently achieving target recycling and amplification. The amplified products of the HP-cDNA duplex were separated rapidly from other DNAs by MCE. Under optimal conditions, the limit of detection of the target DNA was as low as 12.3 pM (S/N = 3). The proposed ISDPR with MCE method was also successfully applied to detect methicillin-resistant S. aureus, and the experimental results showed that it had some advantages such as being label free, ultrasensitive, rapid and well separated.

Investigation of hydroxypropyl-ß-cyclodextrin-based synergistic system with chiral nematic mesoporous silica as chiral stationary phase for enantiomeric separation in microchip electrophoresis.

The separation of chiral amino acids using microchip electrophoresis (MCE) was investigated using chiral nematic mesoporous silica (CNMS) as the chiral stationary phase, with hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as the chiral selector. Individually, neither CNMS nor HP-ß-CD achieved separation, so they were combined. Ten chiral amino acids (phenylalanine, tryptophan, glutamic, alanine, serine, aspartic acid, cysteine, methionine, tyrosine, and histidine) were selected as the model analytes. Under optimized conditions, we achieved baseline separation of six chiral amino acids, and the other four chiral amino acids displayed improved resolution. These results indicate the presence of a synergistic effect between CNMS and HP-ß-CD, showing that the combination of a chiral stationary phase and a chiral additive is a promising approach for enantioseparation using MCE.