Model, Simulation, and Experiments on Moving Exchange Boundary via Ligand and Quantum Dots in Chip Electrophoresis.

Herein, the quench model of the moving exchange boundary (MEB) was first created via a ligand of 5,5'-dithiobis(2-nitro-benzoic acid) (DTNB) and group of 3-mercaptopropionic acid (MPA) capped on QDs, and then the recovery model was formed via MPA and 2-nitro-5-thiobenzoic acid (TNB) capped on QDs. The theory on MEB dynamics and width was developed based on the two reversible models, the simulation was conducted for the illumination of MEB, and the protocol was described for the MEB runs. The experiments revealed that (i) the quench model could be created via DTNB and MPA capped on QDs and the recovery one could be in situ formed via MPA and TNB capped on QDs, showing the feasibility of MEB models; (ii) the simulations on MEB dynamics and width were in coincidence with the theoretic predictions, showing the validity of two models; and (iii) the experiments demonstrated the validity of models, predictions, and simulations. The models and theory have potential for development of a biosensor, nanoparticle characterization, separation science, and an affinity assay of ligand-QDs.

Rare Earth Elements Lanthanum and Praseodymium Adversely Affect Neural and Cardiovascular Development in Zebrafish (Danio rerio).

Increasing rare earth element (REE) mining and refining activities have led to a considerable release of these substances into aquatic environment, yet the knowledge of their impacts on aquatic organisms is still limited. Here, we explored the developmental effects of 16 REEs (concentration ranged from 0.46 to 1000 mg/L) to zebrafish embryos and highlighted the adverse effects of lanthanum (La) and praseodymium (Pr). Among the multiple developmental parameters measured, the significant effects on swimming behavior and cardiac physiology were the most prominent. Transcriptomic analysis of La and Pr at concentrations of 1.1 to 10 mg/L revealed their rather uniform effects at molecular levels. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis revealed that among others, notch, glutamate, and serotonin signaling, as well as cardiac hypertrophy and cardiac muscle contraction, were significantly affected. These changes of neural signaling were consistent with behavior effects observed and supported by neurotransmitter changes and thus provide a reasonable molecular mechanistic explanation. Furthermore, increased DNA damage and apoptotic activity at high concentrations were observed, especially in the heart. They may contribute to explain the observed adverse morphological and physiological outcomes, such as pericardial edema. The effect concentrations observed in the present study were comparable to the concentrations of REE residues at highly contaminated sites (several mg/L), indicating ecotoxicological effects at environmentally relevant concentrations. Overall, the present data help to clarify the potential developmental toxicity of REEs that was not yet fully recognized and thus contribute to their environmental risk assessment.

Proteomics Analysis of Cellular BRS3 Receptor Activation Reveals Potential Mechanism for Signal Transduction and Cell Proliferation.

Bombesin-like receptor 3 (BRS3), an orphan G protein-coupled receptor (GPCR), plays important roles in our biological system while the exact mechanisms behind it are less known. To get insights of the biological effects upon BRS3 activation, we utilized quantitative proteomics approach to explore the dynamic protein profiling during the stimulation by its ligand. At different time points after stimulation with BRS3 surrogate agonist, the protein profiling in BRS3 overexpressed HEK 293 cells BRS3 (HEK 293-BRS3) was analyzed by nano-LC-MS/MS. In total, 1593 cellular proteins were confidently identified and quantified, including 146 proteins dysregulated at multiple time points and 319 proteins only altered at one time point. Data analysis indicated that BRS3 activation could regulate cell death, survival, and protein synthesis, particularly mRNA translation. Key signaling pathways were revealed for BRS3 signal transduction. In particular, 21 of our identified proteins are involved in the rapamycin (mTOR) signaling pathway. The promotion of mTOR was further confirmed through monitoring its indicative targets upon BRS3 activation. Upon the inhibition of mTOR by rapamycin, cell proliferation was dramatically reversed. Our proteomics data collectively demonstrate that BRS3 activation will lead to cascades of signal transduction and promote cell proliferation. The developed strategy might be utilized to discover the roles of other GPCRs and improve our understanding of their unknown functions.

Comparative Proteomic Analysis of Exosomes and Microvesicles in Human Saliva for Lung Cancer.

Extracellular vesicles (EVs) are cell-derived microparticles present in most body fluids, mainly including microvesicles and exosomes. EV-harbored proteins have emerged as novel biomarkers for the diagnosis and prediction of different cancers. We successfully isolated microvesicles and exosomes from human saliva, which were further characterized comprehensively. Salivary EV protein profiling in normal subjects and lung cancer patients was systematically compared through utilizing LC-MS/MS-based label-free quantification. 785 and 910 proteins were identified from salivary exosomes and microvesicles, respectively. According to statistical analysis, 150 and 243 proteins were revealed as dysregulated candidates in exosomes and microvesicles for lung cancer. Among them, 25 and 40 proteins originally from distal organ cells were found in the salivary exosomes and microvesicles of lung cancer patients. In particular, 5 out of 25 and 9 out of 40 are lung-related proteins. Six potential candidates were selected for verification by Western blot, and four of them, namely, BPIFA1, CRNN, MUC5B, and IQGAP, were confirmed either in salivary microvesicles or in exosomes. Our data collectively demonstrate that salivary EVs harbor informative proteins that might be used for the detection of lung cancer through a noninvasive way.

Simple Chip Electrophoresis Titration of Neutralization Boundary with EDTA Photocatalysis for Distance-Based Sensing of Melamine in Dairy Products.

Melamine was sometimes adulterated to dairy products for false protein content increase in developing countries. However, a portable sensor has not been developed for on-spot determination of melamine in dairy products yet. Herein, a distance-based sensor was advanced for the quantification of melamine in dairy products based on chip electrophoretic titration (ET) of moving neutralization boundary (NB) and EDTA photocatalysis. In the chip sensor, EDTA, H2O2, and leucomalachite green (LMG) were added in the anode well. Under UV light, EDTA photocatalyzes H2O2 and colorless LMG as H2O and color malachite green (MG) with one positive charge. When applying an electric field, the MG in the anode well migrated into the channel and was neutralized with the base in the channel, resulting in colorless MG-OH and NB. If the melamine-content dairy sample was added into the EDTA-H2O2-LMG system, H2O2 reacts with melamine, leading to the decrease of MG. Thus, the higher the melamine content in dairy products, the shorter the distance of NB migration under the given time, implying a distance-based sensor of melamine. A series of experiments manifested the validity of ET-NB sensor for detection of melamine. Moreover, the results revealed the numerous merits of ET-NB sensor, such as good selectivity, high sensitivity (LOD down to 0.20 µM for milk and 0.10 µM for infant formula vs the FDA safety limits of 20 µM for milk and 8.0 µM for infant formula), good repeatability and recoveries (87-108% for milk, 90-107% for formula). Particularly, the cell phone-like sensor was portable, simple (no any pretreatment), rapid (within 15 min), as well as low cost, to evaluate the quality of dairy products. The developed sensor has great potential in on-spot detection of melamine in dairy products as well as other analytes, at which we are testing in our lab.

Comparison of antimicrobial peptide purification via free-flow electrophoresis and gel filtration chromatography.

Antimicrobial peptides (AMPs) are usually small and cationic biomolecules with broad-spectrum antimicrobial activities against pathogens. Purifying them from complex samples is essential to study their physiochemical properties. In this work, free-flow zone electrophoresis (FFZE) was utilized to purify AMPs from yeast fermentation broth. Meanwhile, gel filtration chromatography (GFC) was conducted for comparison. The separation efficiency was evaluated by SDS-PAGE analysis of the fractions from both methods. Our results demonstrated as follows: (i) FFZE had more than 30-fold higher processing capacity as compared with GFC; (ii) FFZE could achieve 87% purity and 89% recovery rate while in GFC these parameters were about 93 and 82%, respectively; (iii) the former had ∼2-fold dilution but the latter had ∼13-fold dilution. Furthermore, Tricine-SDS-PAGE, Native-PAGE, and gel IEF were carried out to characterize the purified AMPs. We found that two peptides existed as a pair with the molecular mass of ∼5.5 and 7.0 kDa, while the same pI 7.8. These two peptides were proved to have the antimicrobial activity through the standardized agar diffusion method. Therefore, FFZE could be used to continuously purify AMPs with high bioactivity, which will lead to its wide application in the clinical and pharmaceutical fields.

A stable and convenient protein electrophoresis titration device with bubble removing system.

Moving reaction boundary titration (MRBT) has a potential application to immunoassay and protein content analysis with high selectivity. However, air bubbles often impair the accuracy of MRBT, and the leakage of electrolyte greatly decreases the safety and convenience of electrophoretic titration. Addressing these two issues a reliable MRBT device with modified electrolyte chamber of protein titration was designed. Multiphysics computer simulation was conducted for optimization according to two-phase flow. The single chamber was made of two perpendicular cylinders with different diameters. After placing electrophoretic tube, the resident air in the junction next to the gel could be eliminated by a simple fast electrolyte flow. Removing the electrophoretic tube automatically prevented electrolyte leakage at the junction due to the gravity-induced negative pressure within the chamber. Moreover, the numerical simulation and experiments showed that the improved MRBT device has following advantages: (i) easy and rapid setup of electrophoretic tube within 20 s; (ii) simple and quick bubble dissipates from the chamber of titration within 2 s; (iii) no electrolyte leakage from the two chambers: and (iv) accurate protein titration and safe instrumental operation. The developed technique and apparatus greatly improves the performance of the previous MRBT device, and providing a new route toward practical application.

An innovative ring-shaped electroeluter for high concentration preparative isolation of protein from polyacrylamide gel.

A ring-shaped electroeluter (RSE) was designed for protein recovery from polyacrylamide gel matrix. The RSE was designed in such a way that a ring-shaped well was used to place gel slices and an enrichment well was used to collect eluted protein samples. With HSA as model protein, the electroelution time was less than 30 min with 80% recovery rate, and the concentration of recovered protein was 50 times higher than that of conventional method. The RSE could be reused at least ten times. The developed device makes great advance towards economic electroelution of biomolecules (such as proteins) from gel matrix.

A new pre-column derivatization for valienamine and beta-valienamine using o-phthalaldehyde to determine the epimeric purity by HPLC and application of this method to monitor enzymatic catalyzed synthesis of beta-valienamine.

Valienamine and ß-valienamine are representative C7 N aminocyclitols with significant glycosidase inhibition activity that have been developed as important precursors of drugs for diabetes and lysosomal storage diseases, respectively. The quantitative analysis of these chiral compounds is crucial for asymmetric in vitro biosynthetic processes for converting valienone into valienamine epimers using aminotransferase. Here, we developed an efficient and sensitive method for separation and quantitative analysis of chiral valienamine using reversed-phase high-performance liquid chromatography (HPLC) through o-phthalaldehyde (OPA) pre-column derivatization of the analytes. The epimers were derivatized by OPA in borate buffer (pH 9.0) at room temperature for 30 s, separated on an Eclipse XDB-C18 (5 µm, 4.6 × 150 mm) column, eluted with 22% acetonitrile at 30 °C for 18 min, and detected by a fluorescence detector using 445 nm emission and 340 nm excitation wavelengths. The average resolution of the epimers is 3.86, and the concentration linearity is in the range of 0.02-20 µg/ml. The method proved to be effective, sensitive, and reliable with good intra- and inter-day precision and accuracy, and successfully evaluated the enantiopreference and catalytic capability of the potential aminotransferases on an unnatural prochiral substrate, facilitating the design of an asymmetric biosynthetic route for optically pure valienamine and ß-valienamine.

In-Vial Temperature Gradient Headspace Single Drop Microextraction Designed by Multiphysics Simulation.

Presented herein is a novel headspace single drop microextraction (HS-SDME) based on temperature gradient (TG) for an on-site preconcentration technique of volatile and semivolatile samples. First, an inner vial cap was designed as a cooling device for acceptor droplet in HS-SDME unit to achieve fast and efficient microextraction. Second, for the first time, an in-vial TG was generated between the donor phase in a sample vial at 80 °C and the acceptor droplet under the inner vial cap containing cooling liquid at -20 °C for a TG-HS-SDME. Third, a simple mathematic model and numerical simulations were developed by using heat transfer in fluids, Navier-Stokes and mass balance equations for conditional optimization, and dynamic illumination of the proposed extraction based on COMSOL Multiphysics. Five chlorophenols (CPs) were selected as model analytes to authenticate the proposed method. The comparisons revealed that the simulative results were in good agreement with the quantitative experiments, verifying the design of TG-HS-SDME via the numerical simulation. Under the optimum conditions, the extraction enrichments were improved from 302- to 388-fold within 2 min only, providing 3.5 to 4 times higher enrichment factors as compared to a typical HS-SDME. The simulation indicated that these improvements in the extraction kinetics could be attributed due to the applied temperature gap between the sample matrix and acceptor droplet within the small volume of headspace. Additionally, the experiments demonstrated a good linearity (0.03-100 µg/L, R2 > 0.9986), low limit of detection (7-10 ng/L), and fair repeatability (<5.9% RSD, n = 6). All of the simulative and experimental results indicated the robustness, precision, and usefulness of TG-HS-SDME for trace analyses of analytes in a wide variety of environmental, pharmaceutical, food safety, and forensic samples.

Design of suitable carrier buffer for free-flow zone electrophoresis by charge-to-mass ratio and band broadening analysis.

In this work, charge-to-mass ratio (C/M) and band broadening analyses were combined to provide better guidance for the design of free-flow zone electrophoresis carrier buffer (CB). First, the C/M analyses of hemoglobin and C-phycocyanin (C-PC) under different pH were performed by CLC Protein Workbench software. Second, band dispersion due to the initial bandwidth, diffusion, and hydrodynamic broadening were discussed, respectively. Based on the analyses of the C/M and band broadening, a better guidance for preparation of free-flow zone electrophoresis CB was obtained. Series of experiments were performed to validate the proposed method. The experimental data showed high accordance with our prediction allowing the CB to be prepared easily with our proposed method. To further evaluate this method, C-PC was purified from crude extracts of Spirulina platensis with the selected separation condition. Results showed that C-PC was well separated from other phycobiliproteins that have similar physicochemical properties, and analytical grade product with purity up to 4.5 (A620/A280) was obtained.

Enhancing resolution of free-flow zone electrophoresis via a simple sheath-flow sample injection.

In this work, a simple and novel sheath-flow sample injection method (SFSIM) is introduced to reduce the band broadening of free-flow zone electrophoresis separation in newly developed self-balance free-flow electrophoresis instrument. A needle injector was placed in the center of the separation inlet, into which the BGE and sample solution were pumped simultaneously. BGE formed sheath flow outside the sample stream, resulting in less band broadening related to hydrodynamics and electrodynamics. Hemoglobin and C-phycocyanin were successfully separated by the proposed method in contrast to the poor separation of free-flow electrophoresis with the traditional injection method without sheath flow. About 3.75 times resolution enhancement could be achieved by sheath-flow sample injection method.

A highly efficient three-phase single drop microextraction technique for sample preconcentration.

A highly efficient three-phase single drop microextraction (SDME) method is presented by using an organic-aqueous compound droplet. A coupling microdevice is designed to produce compound droplets in different sizes conveniently. In this way, the volume ratio of organic phase to aqueous phase in a compound droplet can be significantly reduced. Good operability and droplet stability were observed during extraction under vigorous stirring conditions. Five statins were used as model compounds and spiked in river water and human serum samples to evaluate the analytical performance of the proposed method. By using a 1.2 µL toluene-aqueous compound droplet (volume ratio 0.2 : 1), a 350 to 1712 fold enrichment of statins was obtained within 4 minutes. The results indicate that the proposed method is a very rapid and efficient sample pretreatment method, and is promising for automated and high-throughput applications.

Retardation signal for fluorescent determination of total protein content via rapid and sensitive chip moving reaction boundary electrophoretic titration.

A novel concept and theory of moving reaction boundary (MRB) retardation signal (RMRB) was advanced for determination of total protein content via MRB electrophoretic titration (MRBET). The theoretical results revealed that the retardation extent of boundary displacment, viz., the RMRB value, was as a function of protein content. Thus, the RMRB value of a sample could be used to determine its total protein content according to the relevant calibration curve. To demonstrate the concept and theoretical results, a novel microdevice was designed for the relevant experiments of MRBET. The microdevice has 30 identical work cells, each of which is composed of five ultrashort single microchannels (5 mm). In the microdevice, fluorescein isothiocyanate (FITC) was used to denote MRB motion and RMRB value for the first time, the polyacrylamide gel (PAG) containing protein sample was photopolymerized in microchannels, and the MRB was created with acid or alkali and target protein sample. As compared to the classic Kjeldahl method and conventional MRBET performed in glass tube, the developed titration chip has the following merits: good sensitivity (0.3-0.4 µg/mL vs 150-200 µg/mL of protein concentration, 0.6-0.8 ng vs 30-2000 µg of absolute protein content), rapid analysis (20-60 s vs 15-200 min), and portable low-power (15 V vs 200 V).

A new strategy for highly efficient single-drop microextraction with a liquid-gas compound pendant drop.

Herein, a simple assembly was designed via a capillary and a funnel-like cap to achieve liquid-gas compound pendant drop (CPD) microextraction with great convenience. Due to the increased contact area and adhesion force between the capillary tip and the drop, the proposed method provides considerable flexibility in producing CPDs with different air bubble sizes. Four pesticides were chosen as model analytes to evaluate the proposed method. By using a 1 µL chlorobenzene droplet containing a 1 µL air bubble at a stirring rate of 700 rpm, a 70 to 135-fold enrichment of pesticides was obtained within 3.4 minutes. As compared with a typical SDME, the proposed method showed a 2-fold increase of enrichment factors and a 4-fold decrease of extraction time. Improvement of the extraction efficiency could be ascribed to the increased surface area of the droplet, and the thin film phenomena further improved the extraction kinetics through effective agitation. The results indicate that CPD microextraction could serve as a promising sample pretreatment method for automated high-throughput analyses in a wide variety of research areas.

Negative-pressure-induced collector for a self-balance free-flow electrophoresis device.

Uneven flow in free-flow electrophoresis (FFE) with a gravity-induced fraction collector caused by air bubbles in outlets and/or imbalance of the surface tension of collecting tubes would result in a poor separation. To solve these issues, this work describes a novel collector for FFE. The collector is composed of a self-balance unit, multisoft pipe flow controller, fraction collector, and vacuum pump. A negative pressure induced continuous air flow rapidly flowed through the self-balance unit, taking the background electrolyte and samples into the fraction collector. The developed collector has the following advantages: (i) supplying a stable and harmonious hydrodynamic environment in the separation chamber for FFE separation, (ii) effectively preventing background electrolyte and sample flow-back at the outlet of the chamber and improving the resolution, (iii) increasing the preparative scale of the separation, and (iv) simplifying the operation. In addition, the cost of the FFE device was reduced without using a multichannel peristaltic pump for sample collection. Finally, comparative FFE experiments on dyes, proteins, and cells were carried out. It is evident that the new developed collector could overcome the problems inherent in the previous gravity-induced self-balance collector.

Diagnosis and screening of abnormal hemoglobins.

Hemoglobin (Hb) abnormalities, such as thalassemia and structural Hb variants, are among the most prevalent inherited diseases and are associated with significant mortality and morbidity worldwide. However, there were not comprehensive reviews focusing on different clinical analytical techniques, research methods and artificial intelligence (AI) used in clinical screening and research on hemoglobinopathies. Hence the review offers a comprehensive summary of recent advancements and breakthroughs in the detection of aberrant Hbs, research methods and AI uses as well as the present restrictions anddifficulties in hemoglobinopathies. Recent advances in cation exchange high performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), isoelectric focusing (IEF), flow cytometry, mass spectrometry (MS) and polymerase chain reaction (PCR) etc have allowed for the definitive detection by using advanced AIand portable point of care tests (POCT) integrating with smartphone microscopic classification, machine learning (ML) model, complete blood counts (CBC), imaging-based method, speedy immunoassay, and electrochemical-, microfluidic- and sensing-related platforms. In addition, to confirm and validate unidentified and novel Hbs, highly specialized genetic based techniques like PCR, reverse transcribed (RT)-PCR, DNA microarray, sequencing of genomic DNA, and sequencing of RT-PCR amplified globin cDNA of the gene of interest have been used. Hence, adequate utilization and improvement of available diagnostic and screening technologies are important for the control and management of hemoglobinopathies.

High-resolution nucleic acid detection using online polyacrylamide gel electrophoresis platform.

Polyacrylamide gel electrophoresis (PAGE) is one of the most popular techniques for the separation and detection of nucleic acids. However, it requires a complicated detection procedure and offline detection format, which inevitably leads to band broadening and thus compromises the separation resolution. To overcome this problem, we developed an online PAGE (OPAGE) platform by integrating the gel electrophoresis apparatus with the gel imaging system, so as to obviate the need for the complicated detection procedure. Notably, OPAGE enabled the real-time monitoring of the separation process and the immediate imaging of the separation results once the electrophoresis ended. Using a series of synthetic DNAs with different lengths as samples, we demonstrated that the OPAGE platform enhanced 32-64 % of the number of theoretical plates, showed a robust dynamic range of 0.1-12.5 ng/µL, and realized a limit of detection as low as 0.08 ng/µL DNA. Based on our results, we anticipate that the OPAGE platform is a promising alternative to traditional nucleic acid gel electrophoresis for simple and high-resolution detection and quantification and nucleic acid.

A high stable sample loading for analysis of adult alpha-thalassemia via the improved microarray isoelectric focusing of Hb species.

The isoelectric focusing has realized various improvements, including the protocols and creation of mIEF (microcolumn isoelectric focusing) instruments with excellent sensitivity for screening of diabetes and beta thalassemia. However, the problem of manual sample loading and hydration for the mIEF limits the operational capacity for stably detecting and quantitating most abnormal hemoglobin (Hb). Herein, we provided a high stable sample loading protocol for analysis of alpha thalassemia and Hb variants. In contrast to the previous volume of 20 µl, a 100 µl blood sample solution in this protocol was optimized with mixture of 6.4-7.5 and 3-10 pH carrier ampholytes, pI markers and loaded for 30 mins IPG microcolumn hydration. The hydrated microcolumn was then automatically loaded onto the mIEF chip array to which CH3COOH and NH4OH act as anodic and cathodic solutions. Lastly, the IEF was run for 9 mins. Hb H, Barts, A1c, F, A2 and CS were simultaneously separated and focused with higher resolution and sensitivity in quantifying H and Barts as low as 0.6 and 0.5 % respectively. Accordingly, there was an enhanced stability and linearity with a rapid assay time of 45 secs per sample. Moreover, analysis showed a fitting linear relationship with conventional technology at R2 = 0.9803 for H and R2 = 0.9728 for Barts thereby indicating greater accuracy confirmed by the AUC. Hence, the developed protocol could simply be employed for high stable and throughput batch sample loading of hydration, and accurate separation and quantitation of Hb variants for alpha and beta thalassemia.

A temperature-independent model of dual calibration standards for onsite and point-of-care quantification analyses via electrophoresis titration chip.

Electrophoresis titration chip (ETC) is a versatile tool for onsite and point-of-care quantification analyses because it affords naked-eye detection and a straightforward quantification format. However, it is vulnerable to changes in environmental temperature, which regulates the electrophoretic migration by affecting the ion mobility and the target recognition by influencing the enzyme activity. Therefore, the quantification accuracy of the ETC tests was severely compromised. Rather than using the dry bath or heating/cooling units, we proposed a facile model of dual calibration standards (DCS) to mathematically eliminate the effects of temperature on quantification accuracy. To verify our model, we deployed the ETC device at different temperatures ranging from 5 to 40 °C. We further utilized the DCS-ETC to determine the protein content and uric acid concentration in real samples outside the laboratory. All the experimental results showed that our model significantly stabilized the quantification recovery from 35.31-153.44 % to 99.38-103.44 % for protein titration; the recovery of uric acid titration is also stable at 96.25-106.42 %, suggesting the enhanced robustness of the ETC tests. Therefore, DCS-ETC is a field-deployable test that can offer reliable quantification performance without extra equipment for temperature control. We envision that it is promising to be used for onsite applications, including food safety control and disease diagnostics.