A facile, cost-effective, and high-throughput screening method was developed for enzyme-based assays based on Robolid/Microplate (RLMP) platform. The RLMP platform is constructed by immobilizing enzyme on commercial robolids and combining it with a standard 96-well microplate to achieve high-throughput analysis. The initiation and quenching of enzymatic reaction can be performed by simply sandwiching or unsealing the enzyme-immobilized robolids and the sample-containing microplate. This platform enables measurements of multiple target analytes simultaneously based on immobilized enzymatic reactions, with analysis time independent of the number of wells in the microplate. Using urea as the model analyte, we have shown that the RLMP platform exhibits large linear detection range of up to 10 mM, fast analysis time of 30 min/96 samples, as well as good reproducibility and stability. Measurements of urea in human urine and serum samples were performed using the RLMP platform and were compared with the commercial urea test kit. A good correlation was found between the two methods. This study shows that the present RLMP platform has promising prospects for detection of clinical markers and application in disease diagnosis and biochemical analysis.Topics: Biochemical analysis; Cell and Molecular biology; Enzyme kinetics; Materials science; Medicinal chemistry; Proteins;

•Novel CSMS microsphere-based enzyme nanoreactors to analyze low-MW proteins.•Systematically evaluating different approaches to immobilize trypsin in microspheres.•Size-dependent digestion with enhanced peptide-sequence coverage of low-MW proteins demonstrated.•Simplicity and specificity of CSMS microsphere and trypsin-CSMS nanoreactor.Core-shell mesoporous silica (CSMS) microspheres with tunable mesopores in the shell are highly desired in various bioapplications. With novel CSMS microspheres that are synthesized using a convenient two-phase process, we report in this study the analysis of low molecular-weight (MW < 30 kDa) proteins by combining size-exclusion separation and enzyme immobilization. The obtained CSMS microspheres possess uniform diameter (1.3 μm with a shell thickness of 57 nm), large and tunable perpendicular mesopores (7.9 nm), high surface area (55.5 m2/g), large pore volume (0.12 cm3/g) and excellent water dispersibility. The CSMS microsphere-based enzyme nanoreactors have been fabricated by immobilizing trypsin on the pore channels of the CSMS microspheres using either physical absorption or covalent binding via thiol or aldehyde group with a high loading capacity of 11.8–6.1 mg/g. Due to the unique fibrous pore structure, low MW proteins can enter the channels in the shell to interact with immobilized trypsin, followed by analysis of the digestion products using MALDI-TOF MS or electrophoresis (CE) techniques. The properties and analytical performance of different trypsin-immobilized CSMS microspheres has been systematically evaluated. The results show that the peptide-sequence coverage of the smaller protein is enhanced by using trypsin-CSMS microspheres, indicating the size-dependent digestion which results from the size-exclusion interaction of the mesopores against the high-MW proteins. The present study would pave the way for further applications of mesoporous materials in proteome analysis.Download high-res image (164KB)Download full-size image

•A novel method for on-line C-terminal sequencing of peptides has been developed.•Released AAs are sequentially monitored during the entire progress of CPY digestion.•Sequence of a peptide can be determined with just one OGCE run.•The method exhibits high repeatability and efficiency for peptide sequencing.We report a novel method for sequential on-line C-terminal sequencing of peptides, which combines carboxypeptidase Y (CPY) digestion with on-line derivatization and optically gated capillary electrophoresis with laser-induced fluorescence detection (OGCE-LIF). Various factors that may affect the C-terminal sequencing were investigated and optimized. High repeatability of on-line derivatization and the sequential OGCE-LIF assay of amino acids (AAs) was achieved with relative standard deviation (RSD) (n = 20) less than 1.5% and 3.2% for migration time and peak height, respectively. A total of 13 AAs was efficiently separated in the present study, indicating that the method can be used for sequencing of peptides consisting of the 13 AAs studied. Using two synthesized N-terminally blocked peptides as test examples, we show that the present method can on-line monitor the released AAs with a temporal resolution of 50 s during the entire CPY digestion process. The rates of AA release as a function of digestion time were easily measured; thus, the AA sequence of the peptide was determined with just one OGCE assay. Our study indicates the present approach is an effective, reliable, and convenient method for rapid analysis of the C-terminal sequence of peptides, with potential application in peptide analysis and proteome research.

•Development of 2-D SDI theory for capillary electrophoresis.•Demonstration of 2-D SDI theory via systemic experiments.•High efficiency and accuracy of 2-D SDI technique for CE.•Analyses of amino acid and G6PDH reaction mixture revealing versatile SDI tool.We report here theoretical and experimental studies on the sequential diffusion injection (SDI) for CE analysis. Based on the Fick's second law, a theoretical model for two-dimensional (2-D) diffusion has been developed for our SDI system. The 2-D diffusion model has been demonstrated via systematic experimental studies using standard nicotinamide adenine dinucleotide (NADH) as the model analyte. The results show that the dependence of the NADH peak area (corresponding to the injection amount) on the initial sample concentration, the injection time or the capillary-gap distance is consistent with the deduction of the 2-D diffusion model. It is indicated that the 2-D diffusion, both in longitudinal and transverse directions of the capillary, enhances the injection efficiency in comparison to classical concentration diffusion on the plane interface, and improves the accuracy of the sequential injection without any physical disturbance of the capillary inlet. With the insight understanding of the injection mechanism, we have successfully applied the SDI method for sequential CE analysis of amino acids mixture and online assay of the glucose-6-phosphate dehydrogenase-catalyzed reaction. The present study showed that the SDI is a versatile tool for efficient and accurate sequential CE analysis, not only for online monitoring various bioprocesses but also for continuous analyzing complex samples based on capillary electrophoresis.

•We applied packing technique to fabricate CE-based IMER using single particle as the frits.•It takes less than 5 min to fabricate a 5-mm long IMER using the method.•The microreactor exhibits excellent reproducibility and stability for analysis of enzyme reaction.•The method expands the application of capillary electrophoresis for on-line enzyme assay.A novel method using particle-packing technique to fabricate capillary electrophoresis (CE)-based immobilized enzyme reactor (IMER) was accomplished by utilizing perfusive silica single particles as the frits and large-pore beads as the enzyme supports. The fabrication procedure is rapid and simple; the length and enzyme loading amount of the CE-IMERs could be easily adjusted. Performance and feasibility of the CE-IMERs were investigated using on-line trypsin digestion as the model enzyme reaction. High reproducible on-line enzyme assay was demonstrated with RSD less than 4.1% and 3.8% for peak area and migration time of the substrate and product over 100 consecutive runs, respectively. The enzyme can still maintain the activity for at least 10 days, indicating remarkably stability of the CE-IMERs. The CE-IMERs were successfully applied for accurate analysis of trypsin inhibition as well as on-line digestion of standard proteins (myoglobin and BSA). The present method provides a new interesting alternative to open-tubular and monolithic CE-IMERs, thus expands the application of the CE technique for on-line enzyme assay and analysis and characterization of peptides and proteins.

•We reported the sequential MEKC analysis of Ala racemization reaction.•The method provides sequential injection, rapid derivatization and efficient chiral separation.•Both substrate and product enantiomers were automatically monitored during the whole reaction.•The method exhibits good reproducibility and accuracy for analysis of racemization reaction.A novel method for online monitoring racemization reaction of alanine (Ala) enantiomers was developed, by combining sequential sample injection and micellar electrokinetic chromatography (MEKC) technique. Various conditions were investigated to optimize the sequential injection, Ala derivatization and MEKC chiral separation of d-/l-Ala. High reproducibility of the sequential MEKC analysis was demonstrated by analyzing the standard Ala samples, with relative standard deviation values (n = 20) of 1.35%, 1.98%, and 1.09% for peak height, peak area and migration time, respectively. Ala racemization was automatically monitored every 40 s from the beginning to the end of the reaction, by simultaneous detection of the consumption of the substrate enantiomer and the formation of the product enantiomer. The Michaelis constants of the racemization reaction were obtained by the sequential MEKC method, and were in good agreement with those obtained by traditional off-line enzyme assay. Our study indicated that the present sequential MEKC method can perform fast, efficient, accurate and reproducible analysis of racemization reaction of amino acids, which is of great importance for the determination of the activity of racemase and thus understanding its metabolic functions.

A novel capillary electrophoresis (CE)-based immobilized enzyme reactor (IMER) using graphene oxide (GO) as a support was developed by using a simple and reliable immobilization procedure based on layer by layer electrostatic assembly. Using trypsin as a model enzyme, the performance of the fabricated CE-based IMERs was evaluated. Various conditions, including trypsin concentration, trypsin coating time, number of trypsin layers and buffer pH, were investigated and optimized. The Michaelis constant Km (0.24 ± 0.02 mM) and the maximum velocity Vmax (0.32 ± 0.04 mM s−1) were determined using the CE-based IMERs, and the values are consistent with those obtained using free trypsin, indicating that enzyme immobilized via the proposed approach does not cause a significant structural change of the enzyme or any reduction of enzyme activity. The presented CE-based IMERs exhibit excellent reproducibility with RSD less than 2.8% over 20 runs, and still remain 79.5% of the initial activity after five days with more than 100 runs. Using the proposed CE-based IMERs, the digestion of angiotensin was completed within 3 min, while quite a number of trypstic peptides were observed for BSA on-line digestion with an incubation time of 30 min. As identified by MS analysis, the online digestion products of BSA using the present CE-based IMER are comparable with those obtained using free trypsin digestion for 12 h incubation. It is indicated that the present immobilization strategy using GO as a support is reliable and practicable for accurate on-line analysis and characterization of peptides and proteins.

We reported the application of graphene as a novel stationary phase in open-tubular capillary electrochromatography (OTCEC). The OT column was easily fabricated by electrostatic assembly of poly(diallydimethylammonium chloride) followed by self-adsorption of negatively charged graphene. The chromatographic properties of graphene-coated OT column were investigated by separation of nitroaniline isomers through variation of the buffer pH, sodium dodecyl sulfate concentration and separation voltage. The results demonstrate the application of graphene in OTCEC greatly improves the separation by largely enhancing the surface area thus increasing stationary/mobile phase interaction. The proposed OT column exhibits good repeatability with run-to-run RSD of retention time as low as 1.5% (n = 5), and excellent stability for at least two-week usage with total ∼200 runs. The method was successfully applied for analysis of nitroaniline isomers in hair dye samples.Highlights► We reported the application of graphene as a novel stationary phase in OTCEC. ► We investigated the chromatographic properties of graphene-coated OT column. ► Application of graphene in OTCEC greatly improves the separation efficiency. ► Graphene-coated OT column exhibits good repeatability and excellent stability. ► The method was successfully applied for separation of neutral explosive samples.

•A cyclodextrin-modified gold-nanoparticles is demonstrated as chiral stationary phase in monolith CEC.•The performance of enantioselective monolithic column was investigated.•Enantioselective monolithic column exhibits good reproducibility and excellent stability.•The method was successfully applied for chiral separation of drug enantiomers.β-cyclodextrin modified gold nanoparticles (CD-GNP) were employed as the stationary phase in monolith capillary electrochromatography (CEC) to facilitate enantioseparation. CD-GNP were covalently bound to the surface of the thiolated porous polymer monolithic column. The fabricated enantioselective monolithic column was characterized by a variety of spectroscopic methods. The column exhibited steady EOF mobility over pH values ranging from 4.6 to 9.7. Additionally, the column was stable under CEC separation conditions over 180 min. Moreover, the column exhibited good column-to-column reproducibility. The CD-GNP-modified monolithic column was employed in the efficient CEC separation of three pairs of drug enantiomers (chlorpheniramine, zopiclone and tropicamide). The results exhibit reproducible run-to-run enantioseparations and the monolith column can maintain its enantioselectivity for more than 1 month if the column is stored in a CD-GNP solution at 4 °C.

We proposed the first application of an electrophoretically mediated microanalysis (EMMA) method for fast online discrimination and determination of substrate enantiomers, which was achieved by just one EMMA assay. Lactate dehydrogenase (LDH)-catalyzed reaction was studied to evaluate the feasibility and performance of the presented method. The l- and d-LDH chiral enzymatic reactions, which are highly stereoselective to the lactate enantiomers, were initiated successively in one capillary, and the corresponding products, nicotinamide adenine dinucleotide (NADH), were online discriminated and detected by UV absorption. Excellent linear dependence of the two NADH peak intensities on the concentration of the corresponding lactate enantiomers was obtained within a wide range of 0.1–10 mM. The limit of detection was 26 μM for d-lactate and 49 μM for l-lactate (S/N = 3). Good repeatability of online chiral discrimination was demonstrated with relative standard deviation (RSD) < 6.3% for NADH peak height and RSD < 1.5% for migration time (n = 5). Km values for l- and d-lactate were measured and were consistent with those of the off-line enzyme assays. The presented method was successfully applied to determine the l-/d-lactate in several yogurt and wine samples. Our study shows a new application of the EMMA method utilizing high stereoselectivity of enzymes for fast online chiral analysis.

We have developed an easy-to-operate and effective method for performing the sequential online analysis of enzyme reactions based on capillary electrophoresis (CE). The system was constructed by passing two capillaries through a sample vial at a distance of 5 μm between the capillary ends. Direct online sample injection and sequential CE analysis were achieved by periodically switching the high-voltage power supply off and on, without any physical disturbance of the capillary inlet. The sample was injected via concentration diffusion with in-column derivatization of the amino acids occurring at the interface of the capillaries. High reproducibility of the sequential injections was demonstrated with relative standard deviation values (n = 20) of 1.01%, 1.25%, and 0.80% for peak height, peak area, and migration time, respectively. Sequential online CE enzyme assay of a glutamate pyruvate transaminase catalyzed enzyme reaction was carried out by simultaneously monitoring the substrate consumption and the product formation every 30 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. Our method has great potential for usage in sequential online CE analysis of chemical reactions with in-column chemical derivatization of the analytes for ultraviolet or laser-induced fluorescence detection.

Enantioselective open-tubular CEC (OTCEC) with thiolated β-CD modified gold nanoparticles (CD-GNPs) as stationary phase was developed. The enantioselective OT capillary column was fabricated by electrostatic assembly of poly(diallydimethylammonium chloride) (PDDA) followed by self-adsorption of negatively charged CD-GNPs. The enantioselective capillary column has a steady EOF mobility over a wide pH range of 3.0 to 9.2 (RSD 4.8%), and is quite stable over 240 min with very good column to column reproducibility. Efficient enantioseparation of the presented method was demonstrated by analyzing three drug enantiomers. Our results show that the column exhibits good run-to-run repeatability for enantioseparations and can maintain the enantioselectivity for more than 1 month if the column was stored in CD-GNPs solution at 4 °C.

A facile and fast microwave irradiation method was developed to prepare PtPd bimetallic alloy nanoparticles on onion-like mesoporous carbon vesicle (MCV). With MCV acts as a template, its high surface area favors the formation of nanosized PtPd particles. The PtPd/MCV nanocomposite was characterized by transmission electron microscopy (TEM), scanning electron micrographs (SEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). A nonenzymatic amperometric sensor of glucose based on the PtPd/MCV modified glassy carbon (GC) electrode is developed. Compared with the Pt/MCV nanocomposite, the PtPd/MCV modified electrode displays enhanced current response towards glucose and gives linear range from 1.5 to 12 mM. The particular lamellar structure of the MCV results in favorable transport passage for glucose. The modified electrode achieves 95% of the steady-current within 3 s. This nonenzymatic glucose sensor also exhibits good ability of anti-interference to electroactive molecules. The fast response and facile preparation method make PtPd/MCV nanocomposite promising for the development of enzyme-free sensor for glucose.

A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5 s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.