The sensitive detection of biomarkers is crucial for the early identification and treatment of cancer. In this study, a cysteine-histidine-Cu-modified jujube-like Cu2O (CH-Cu@J-Cu2O) nanozyme was synthesized and used to fabricate an electrochemical sensor for mucin-1 (MUC1) sensitive detection. Gold-modified reduced graphene oxide and CH-Cu@J-Cu2O for the sensor were prepared and also characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). The CH-Cu@J-Cu2O nanozyme was used as a signal probe, containing two catalytic units of cysteine- histidine-Cu and jujube-like Cu2O. The CH-Cu@J-Cu2O nanozyme can potently catalyze the H2O2-driven oxidation of dopamine to aminochrome, leading to a high-level electrochemical signal. This electrochemical sensor was used to detect MUC1 with a linear range from 0.5 to 5000 pg.mL(-1), and the limit of detection was 0.085 pg.mL(-1), owing to the excellent catalytic activity of CH-Cu@J-Cu2O. The expression of MUC1 on the surface of MCF-7 cells was further analyzed, and the results indicate that the proposed strategy is practical for the detection of biomarkers.

Abstract 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.

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.

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.

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.

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.

Probe-lengthening amplification (PLA) is a target-specific nucleic acid amplification method that realizes good selectivity by effectively avoiding nonspecific amplification. Here, we propose a probe-lengthening amplification-assisted microchip electrophoresis (MCE) strategy for sensitive analysis of 16S rRNA genes of five bacteria. In this assay, four specific short probes were designed for a target bacterium to recognize its bacterial 16S rRNA gene, integrated into longer DNA ligation duplexes using Ampligase, and subsequently separated and detected by MCE. Along with the rapid generation of ligation duplexes, this approach provides exponential amplification of nucleic acid signals that are useful for sensitive bacterial quantification. Through tactfully combining PLA and MCE, the detection sensitivity of bacterial genes was significantly improved, and a limit of detection (LOD) of 30 fM was realized for the artificial target DNA. This approach was also applied to detect actual bacterial genomic samples with excellent results, demonstrating the potential application of this methodology in infection diagnosis.

Because foodborne pathogenic bacteria are in a great variety and may cause many infectious diseases even at low concentrations, a highly sensitive and selective method has long-been desired for bacteria detection. In this study, a microchip electrophoretic method for biosensing E. coli O157:H7 was developed by using E. coli O157:H7 aptamer (apt-E) for specific bacteria recognition together with aptamer-induced catalysed hairpin assembly (CHA) for significantly improving the sensitivity of bacteria detection. Briefly, three nucleic acid strands (apt-E, hairpin H1, and H2) were used in the CHA amplification. Because different quantities of H1/H2 complexes were formed due to the circle amplification induced with different amounts of apt-E and the correlation between the concentrations of apt-E and E. coli O157:H7, E. coli O157:H7 thus could be quantified by the detection of H1/H2 complexes with microchip electrophoresis (MCE). Under the optimal conditions, the limit of detection was 75 CFU mL(-1). This method was also applied to detect E. coli O157:H7 in defatted milk with a satisfying recovery rate. The proposed strategy for E. coli O157:H7 detection is label-free, enzyme-free, ultrasensitive, and cost-effective. It is also practical and could be applied to detect other bacteria in food samples.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main pathogens involved in hospital and com-munity 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.

The specific and sensitive detection of multiple pathogens is critical for the prevention and identification of health- and safety-related problems. A microchip electrophoresis/LED-induced fluorescence (MCE-LIF) method, combining an aptamer-based probe and a novel universal primer-duplex polymerase chain reaction (PCR) process (UP-DPCR), was designed to simultaneously detect two kinds of bacteria. The probe consists of a recognition unit (aptamer) for specifically capturing bacterial cells and eventually releasing complementary DNAs (C1 and C2). The two released DNA strands (C1 and C2) can be simultaneously amplified by a pair of universal primers, because of the identical sequences designed at both ends of the two DNA strands. The UP-DPCR products of C1 and C2 can be separated and detected by MCE-LIF, and the heights of the two peaks are correlated with the concentrations of the corresponding bacteria. Here, Salmonella enterica serovar Typhimurium (S. Typhimurium) and Pseudomonas aeruginosa (P. aeruginosa) were detected as a proof of concept. Under optimal conditions, the limits of detection (S/N = 3) were 15 CFU mL(-1) for S. Typhimurium and 5 CFU mL(-1) for P. aeruginosa. This approach was also applied for detecting these two types of bacteria in defatted milk, indicating its potential application in the analysis of real samples. This method can not only simultaneously detect two kinds of bacteria without lysing the bacterial cells, but also simplify duplex PCR with the use of universal primers. (C) 2019 Elsevier B.V. All rights reserved.

Bacterial lipopolysaccharides (LPS) are endotoxins, which can cause fever, inflammation, cell and tissue damage, irreversible septic shock and death in mammals as well as other organisms. Herein, a highly sensitive and selective strategy was proposed for detecting LPS using microchip electrophoresis (MCE) based on aptamer-modified magnetic beads and polymerase chain amplification. In this strategy, the biotinylated aptamer for the LPS (LBA) was hybridized partially with the complementary DNA (cDNA), then the double-stranded DNA obtained was immobilized onto the magnetic beads. In the presence of target LPS, the target-aptamer complex was formed and the cDNA would be freed. After a magnetic separation, the cDNA was released into the supernatant and subsequently triggered the polymerase chain amplification. This process eventually generated a large number of output DNA, which could be quantitatively detected by MCE. With the polymerase chain amplification, this designed protocol provided an ultrasensitive detection of LPS down to the 1.1 x 10(-14) g/mL with a linear range of 5 orders of magnitude. The proposed method also exhibited a high specificity toward LPS in the presence of other common interfering substances. Moreover, the assay showed a good practical application for LPS determination in serum samples.

该文在水相溶剂中绿色合成了γ-氨基丁酸键合硅胶(Sil-GABA)色谱固定相,采用红外光谱、元素分析等对其进行了表征,并将制备的Sil-GABA材料作为毛细管色谱柱的填料研究了其色谱分离模式和分析性能.结果 显示,在以乙酸铵水溶液为流动相的条件下,该填料可有效分离硝基氯苯3种位置异构体和7种磺胺,表明Sil-GABA固定相具有富水作用色谱的特性;在高浓度有机流动相条件下可有效分离4种苯甲酸类化合物,表明Sil-GABA具有亲水作用色谱的特性;此外,该Sil-GABA固定相在反相模式下对硝基苯胺异构体也有一定的分离能力.在优化条件下,7种磺胺保留时间的相对标准偏差小于3.2％,表明制得的色谱柱具有良好的重复性.该Sil-GABA固定相具有多种保留机理,在毛细管液相色谱分离领域具有潜在的应用价值.

An aptamer based assay is described for the determination of Salmonella typhimurium (S.typhimurium). A metal-organic framework-graphene composite of type UiO-67/GR is used as the substrate, and an aptamer-gold nanoparticles-horseradish peroxidase (Apt-AuNP-HRP) conjugate the signal amplification probe. A phosphate-terminal and partially complementary DNA (cDNA) of the aptamer is covalently bound to UiO-67/GR via the chemical complexation between phosphate and Zr-OH groups of UiO-67, and then S. typhimurium and cDNA will compete for the binding sites. The binding of Apt-AuNP-HRP to S.typhimurium leads to the formation of strong conjugates. The unbound signal probes then attach to the surface of a glassy carbon electrode via hybridization with cDNA. This generates a large current response (best measured at a potential as low as −0.02 V vs. saturated calomel electrode) under the catalytic action of HRP on the H2O2-hydroquinone system. Under the optimal conditions, the differential pulse voltammetric signal decreases linearly in the 2 × 101 – 2 × 108 cfu·mL−1 S.typhimurium concentration range, with a lower detection limit of 5 cfu·mL−1 (based on S/N = 3). The method was successfully applied to the detection of S. typhimurium in spiked milk samples.

In this study, a mixed mode stationary phase for capillary liquid chromatography (cLC) was prepared by chemical modification of glycol diglycidyl ether (EGDE) and γ-aminobutyric acid (GABA) bonded to the silica, named as Sil–EGDE–GABA. The Sil–EGDE–GABA was characterized by Fourier transform infrared spectroscopy and elemental analysis. The retention of nucleotide bases at different acetonitrile levels in the mobile phase indicated that the stationary phase possessed both hydrophilic chromatography (HILIC) and reversed-phase chromatography (RPLC) characteristics. Positional isomers, aniline compounds and a mixture of polar–nonpolar analytes were separated in RPLC mode. The stationary phase exhibited different separation mechanisms in the separation of positional isomers, compared with a commercial ODS column. Sulfonamides and biogenic amines were successfully separated in HILIC mode. These results demonstrated the application possibilities of the prepared Sil–EGDE–GABA as packing materials in cLC.

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.

The detection of Salmonella enterica serovar Typhimurium (S. Typhimurium) is very important for the prevention of food poisoning and other infectious diseases. Here we reported a simple and sensitive strategy to test S. Typhimurium by microchip capillary electrophoresis couple with laser-induced fluorescence (MCE-LIF) based on the specific reaction between the bacterium and corresponding aptamers. Based on the differences in charge to mass ratio between bacteria-aptamer complexes and free aptamers, a separation of the complexes and free aptamers could be obtained by MCE. The optimal parameters of the specific reaction including fluorescent dye concentration, Mg2+ concentration, incubation time, and pH of incubation solution were carefully investigated. Meanwhile, a non-specific DNA was exploited as a contrast for the detection of S. Typhimurium. Under the optimal conditions, a rapid separation of the bacteria-aptamer complex and free aptamers was achieved within 135 s with a limit of detection (S/N =3) of 3.37 x 10(2) CFU mL(-1). This method was applied for the detection of S. Typhimurium in fresh milk samples and a recovery rate of 95.8% was obtained. The experimental results indicated that the specific aptamers are of enough biostability and the established method could be used to analyze S. Typhimurium in foods. (C) 2017 Elsevier B.V. All rights reserved.