Deciphering Nucleic Acid Binding Proteome of Mouse Immune Organs Reveals Hub Proteins for Aging.

Profiling the nucleic acid-binding proteins (NABPs) during aging process is critical to elucidate its roles in biological systems as well as transcriptional and translational regulation. Here, we developed a comprehensive strategy to survey the NABPs of mouse immune organs by using single cell preparation and selective capture technology-based proteomics. Our approach provided a global view of tissue NABPs from different organs under normal physiological conditions with extraction specificity of 70 to 90%. Through quantitative proteomics analysis of mouse spleen and thymus at 1, 4, 12, 24, 48, and 72 weeks, we investigated the molecular features of aging-related NABPs. A total of 2674 proteins were quantified in all six stages, demonstrating distinct and time-specific expression pattern of NABPs. Thymus and spleen exhibited unique aging signatures, and differential proteins and pathways were enriched across the mouse lifespan. Three core modules and 16 hub proteins associated with aging were revealed through weighted gene correlation network analysis. Significant candidates were screened for immunoassay verification, and six hub proteins were confirmed. The integrated strategy pertains the capability to decipher the dynamic functions of NABPs in aging physiology and benefit further mechanism research.

A facile double moving redox boundary model for visual electrophoresis titration of ascorbic acid.

In this work, we proposed a double moving redox boundary (MROB) model to realize the colorless analyte electrophoresis titration (ET) by the two steps of the redox reaction. Single MROB has been proposed for the development of ET sensing (Analyst, 2013, 138, 1137. ACS Sensor, 2019, 4, 126.), and faces great challenges in detecting the analyte without color change during redox reaction. Herein, a novel model of double-MROB electrophoresis, including its mechanisms, equations, and procedures, was developed for titration by using ascorbic acid as a model analyte. The first MROB was created with ferric iron (Fe3+) and iodide ion (I-) in which Fe3+ was reduced as Fe2+ and I- was oxidized as molecular iodine (I2) used as an indicator of visible MROB due to blue starch-iodine complex. The second boundary was then formed between the molecular iodine and model analyte of ascorbic acid. Under given conditions, there was a quantitative relationship between velocity of MROB (VMROB(ii)) and ascorbic acid concentration (CVit C) in the double-MROB system (1/VMROB(ii) = 0.6502CVit C + 4.5165, and R = 0.9939). The relevant relative standard deviation values of intraday and inter-day were less than ∼5.55% and ∼6.64%, respectively. Finally, the titration of ascorbic acid in chewable vitamin C tablets was performed by the developed method, the titration results agreed with those via the classic iodometric titration. All the results briefly demonstrated the validity of the double MROB model, in which Vit C was used as a model analyte. The developed method had potential use in quantitative analysis of redox-active species in biomedical samples.

Enrichment of low-abundance osteopontin in bovine milk via reciprocating free-flow isoelectric focusing.

Osteopontin (OPN) in milk plays an important role in intestinal and brain development in early infancy, and great attention has been focused on OPN isolation to add extra OPN in infant formula. However, large-scale OPN isolation is limited by the low efficiency of sample pretreatment. Herein, we utilized preparative reciprocating free-flow isoelectric focusing (RFFIEF) to showcase the enrichment of low-abundance OPN in bovine milk, which contained an extremely high concentration of unwanted proteins. The reciprocating IEF format and the design of the multi-channel collector allowed us to enrich OPN in 1 L milk within 6 h. We removed 97.5% of unwanted proteins and obtained an enrichment factor of 11. Thus, our RFFIEF method can be applied to the preparative pretreatment of the large-scale milk sample and potentially improve the efficiency of downstream OPN purification.

Marker-Free Isoelectric Focusing Patterns for Identification of Meat Samples via Deep Learning.

Isoelectric focusing (IEF) is a powerful tool for resolving complex protein samples, which generates IEF patterns consisting of multiplex analyte bands. However, the interpretation of IEF patterns requires the careful selection of isoelectric point (pI) markers for profiling the pH gradient and a trivial process of pI labeling, resulting in low IEF efficiency. Here, we for the first time proposed a marker-free IEF method for the efficient and accurate classification of IEF patterns by using a convolutional neural network (CNN) model. To verify our method, we identified 21 meat samples whose IEF patterns comprised different bands of meat hemoglobin, myoglobin, and their oxygen-binding variants but no pI marker. Thanks to the high throughput and short assay time of the microstrip IEF, we efficiently collected 1449 IEF patterns to construct the data set for model training. Despite the absence of pI markers, we experimentally introduced the severe pH gradient drift into 189 IEF patterns in the data set, thereby omitting the need for profiling the pH gradient. To enhance the model robustness, we further employed data augmentation during the model training to mimic pH gradient drift. With the advantages of simple preprocessing, a rapid inference of 50 ms, and a high accuracy of 97.1%, the CNN model outperformed the traditional algorithm for simultaneously identifying meat species and cuts of meat of 105 IEF patterns, suggesting its great potential of being combined with microstrip IEF for large-scale IEF analyses of complicated protein samples.

Quadruple UV LED Array for Facile, Portable, and Online Intrinsic Fluorescent Imaging of Protein in a Whole Gel Electrophoresis Chip.

Intrinsic fluorescence imaging (IFI) has been used for the stain-free detection of proteins in slab gel. However, complicated detection setups and small irradiation area limited the development of facile, online, and portable imaging of the whole slab gel. We here designed a quadruple UV LED array to produce even and powerful area light for direct irradiation of gel electrophoresis chip (GEC) at 275 nm. In addition, we only used a filter of 365 nm, a UV camera lens, and a CCD for IFI detection. We integrated the simple detection setup with the small GEC to construct the IFI-GEC device with a portable size of 15 × 15 × 38 cm. We detected three model proteins to demonstrate the good evenness of the LED array and the online imaging of the whole GEC. Furthermore, the reproducible IFI-GEC detection was completed within 10 min and the LOD was as low as 40 ng for lysozyme detection. All results indicated the potential of the IFI-GEC device for online and portable detection of proteins without staining.

Analyses of oxidative DNA damage among coal vendors via single cell gel electrophoresis and quantification of 8-hydroxy-2'-deoxyguanosine.

Looking at the development status of Nigeria and other developing nations, most low-income and rural households often use coal as a source of energy which necessitates its trade very close to the communities. Moreover, the effects of exposure to coal mining activities are rarely explored or yet to be studied, not to mention the numerous street coal vendors in Nigeria. This study investigated the oxidative stress levels in serum and urine through the biomarker 8-OHdG and DNA damage via single cell gel electrophoresis (alkaline comet assay). Blood and urine levels of 8-OHdG from 130 coal vendors and 130 population-based controls were determined by ELISA. Alkaline comet assay was also performed on white blood cells for DNA damage. The average values of 8-OHdG in serum and urine of coal vendors were 22.82 and 16.03 ng/ml respectively, which were significantly greater than those detected in controls (p < 0.001; 15.46 and 10.40 ng/ml of 8-OHdG in serum and urine respectively). The average tail length, % DNA in tail and olive tail moment were 25.06 µm, 18.71% and 4.42 respectively for coal vendors. However, for controls, the average values were 4.72 µm, 3.63% and 1.50 for tail length, % DNA in tail and olive tail moment respectively which were much lower than coal vendors (p < 0.001). Therefore, prolonged exposure to coal dusts could lead to higher serum and urinary 8-OHdG and significant DNA damage in coal vendors observed in tail length, % DNA in tail, and olive tail moment by single cell gel electrophoresis. It is therefore established that coal vendors exhibit a huge risk from oxidative stress and assessment of 8-OHdG with single cell gel electrophoresis has proven to be a feasible tool as biomarkers of DNA damage.

Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins.

Cellular nucleic acid-binding proteins (NABPs), namely, DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in many biological processes. However, extracting NABPs with high efficiency in living cells is challenging, which greatly limited their proteomics analysis and comprehensive characterization. Here, we discovered that titanium (IV) ion-immobilized metal affinity chromatography (Ti4+-IMAC) material could enrich DNA and RNA with high efficiency (96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore developed a Ti4+-IMAC method for the joint extraction of DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking, DBPs and RBPs were covalently linked to nucleic acids (NAs) and further denatured by organic solvents. After Ti4+-IMAC capture, 2000 proteins were identified in 293T cells, among which 417 DBPs and 999 RBPs were revealed, showing promising selectivity for NABPs. We further applied the Ti4+-IMAC capture method to lung cancer cell lines 95C and 95D, which have different tumor progression abilities. The DNA- and RNA-binding capabilities of many proteins have been dysregulated in 95D. Under our conditions, Ti4+-IMAC can be used as a selective and powerful tool for the comprehensive characterization of both DBPs and RBPs, which might be utilized to study their dynamic interactions with nucleic acids.

Gel Electrophoresis Chip Using Joule Heat Self-Dissipation, Short Run Time, and Online Dynamic Imaging.

Gel electrophoresis (GE) is one of the most general tools in biomedicine. However, it suffers from low resolution, and its mechanism has not been fully revealed yet. Herein, we presented the dispersion model of w2 (t) ∝ Tt, showing the band dispersion (w) via temperature (T) and running time (t) control. Second, we designed an efficient GE chip via the time control and rapid Joule heat self-dissipation by thermal conductive plastic (TCP) and electrode buffer. Third, we conducted the simulations on TCP and polymethylmethacrylate (PMMA) chips, unveiling that (i) the temperature of TCP was lower than the PMMA one, (ii) the temperature uniformity of TCP was better than the PMMA one, and (iii) the resolution of TCP was superior to the PMMA one. Fourth, we designed both TCP and PMMA chips for experimentally validating the dispersion model, TCP chip, and simulations. Finally, we applied the TCP chip to thalassemia and model urine protein assays. The TCP chip has merits of high resolution, rapid run of 6-10 min, and low cost. This work paves the way for greatly improving electrophoretic techniques in gel, chip, and capillary via temperature and time control for biologic study, biopharma quality control, clinical diagnosis, and so on.

A facile online multi-gear capacitively coupled contactless conductivity detector for an automatic and wide range monitoring of high salt in HPLC.

Sensing the electrolyte solution or aqueous-organic mixture has attracted much interest in chemical separation, pharmaceutical engineering, bioprocess, and biochemical experiments. However, reports on online contactless sensor with automatic and wide range sensing of high content electrolyte have been rarely presented. Herein, a facile model and theory of online multi-gear capacitively coupled contactless conductivity detection (M-C4D) sensor was proposed using one excitation electrode and multiple detection electrodes. Further, the relevant digital computation based on the M-C4D theory was developed for parameter optimization: the electrode gap of 5-150 mm, inner radius of 0.25-0.75 mm, electrode length of 10-60 mm, and frequency of 40-250 kHz using MATLAB. To demonstrate the model, theory, and digital computation, liquid chromatography (LC) was chosen as the model of bioprocess, and the sensor was designed and used as an online sensing device for the automatic monitoring of high salt elution in LC. The experiments showed that (i) the detection results were in agreement with the digital data, validating the digital computation, theory, and model of M-C4D and (ii) the monitoring data of M-C4D were in agreement with those via the traditional meter, further validating the model and theory. Finally, the developed sensor was applied to the automated detection of high salt gradient in LC. In contrast to the currently used meters and C4D, the developed M-C4D sensor had the following merits: (i) facile and automatic online detection avoiding cumbersome manual switching of detector heads, (ii) fair linear range of 0.015-20 mS cm-1 (equivalently 0.1-159 mM KCl) that does not fit the range of traditional C4D, and (iii) fair accuracy of less than 1.50% relative error. All these results indicate that the developed model, theory, and sensor have potential for the process monitoring of high content electrolytes transfer in biochemical engineering and clinic pre-warning.

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.

Metal Organic Framework Nanomaterial-Based Extraction and Proteome Analysis of Membrane and Membrane-Associated Proteins.

Membrane proteins (MPs) play a key role in various biological processes, while difficulties still exist in the extraction because of their inherent low abundance and poor solubility caused by high hydrophobicity. Metal organic framework (MOF) materials with good hydrophobic properties have the ability to absorb MPs, especially zeolitic imidazolate framework (ZIF) materials. Here, two MOF materials (ZIF-8 and ZIF-67) were compared for MP extraction, and our results revealed that higher yield was obtained with ZIF-67. After method development, the optimal enrichment effect was obtained when the mass ratio of proteins and ZIF-67 reached 1:20 with 100 mM NaCl in 20% ethanol at 4 °C and pH 9.0. When compared with a commercial kit, the extraction yield increased by 88.11% and the average number of identified MPs elevated by 29.17% with the developed ZIF method. Normal lung cell MRC5 was employed to verify the effectiveness of the ZIF method. Results showed 45.13% increase in yield and 22.88% increase in average number of identified MPs by the ZIF method. Our method was further applied to the enrichment of MPs for high-metastatic (95D) and low-metastatic (95C) human lung cancer cells. A total of 1732 (95D) and 1711 (95C) MPs were identified, among which 710 MPs were dysregulated significantly; 441 upregulated MPs in 95D cells were found to be closely related to the growth, proliferation, and migration of lung cancer cells. Our results collectively demonstrated that ZIF-67 was an ideal material for MP extraction, which might be helpful for analysis of cancer proteomics and discovery of cancer migration associated MPs.

Cancer Cell Derived Small Extracellular Vesicles Contribute to Recipient Cell Metastasis Through Promoting HGF/c-Met Pathway.

Cancer progression is frequently caused by metastasis and leads to significantly increased mortality. Cell derived extracellular vesicles, including exosomes, in the microenvironment play key roles in cellular signal transduction, whereas their biological function in cancer metastasis and progression needs in-depth investigation. Here, we initially demonstrate that the small extracellular vesicles (sEVs) derived from highly metastatic lung cancer cells exhibited great capacity to promote the progression of recipient cells. Quantitative proteomics was employed to comprehensively decipher the proteome of cell derived sEVs and more than 1400 sEVs proteins were identified. Comparison analysis indicates that sEVs-HGF is a potential metastasis related protein and our verification data from clinical lung cancer plasma samples and in vivo experiments further confirmed the association. We found that sEVs-HGF could induce epithelial-mesenchymal transition and the coordination between HGF and c-Met was confirmed through corresponding target knockdown and kinase inhibition. Our data collectively demonstrate that cancer cell derived sEVs contribute to recipient cell metastasis through promoting HGF/c-Met pathway, which are potential targets for the prevention and treatment of cancer metastasis.

Free-Flow Isoelectric Focusing for Comprehensive Separation and Analysis of Human Salivary Microbiome for Lung Cancer.

Human microbiome contains billions of microorganisms that play important roles in the biological system and different diseases. Due to its complexity, conventional culture-independent technology may underestimate the value of low-abundance bacteria, which calls for a highly efficient method for its enrichment and comprehensive analysis. In this study, we developed a recycling free-flow isoelectric focusing (RFFIEF) method-based electrophoresis method to separate salivary microbiome. First, we used Escherichia coli (DH5α) as a model for RFFIEF method development, which was focused in a narrow pH range (0.38 pH unit). The recovery rate was 80.81% with 5.85% relative standard deviation (n = 5). The optimized method was then adopted to separate the human salivary microbiome into 32 fractions, followed by 16S rRNA gene sequencing and metaproteomics analysis. After RFFIEF fractionation, we identified 508 bacterial genera, which increased by 225% on average (n = 3) when compared to the results before fractionation. We further compared the compositional change of microbiome in the saliva of lung cancer group (n = 22) and control group (n = 21) through RFFIEF. Quantitative results demonstrated that six bacterial genera were upregulated dramatically in the lung cancer group, while two genera were downregulated. Through qPCR verification in an independent sample set (n = 48), we confirmed that genus Granulicatella was significantly upregulated in the lung cancer group, whereas Pseudomonas was remarkably downregulated (p < 0.001). RFFIEF is an efficient and reproducible technology to fractionate the microbiome for its comprehensive analysis, which can be further applied to the in-depth study of the complex microbiomes and contribute to the discovery of disease-associated bacteria.

Purification of low-abundance lysozyme in egg white via free-flow electrophoresis with gel-filtration chromatography.

As an effective separation tool, free-flow electrophoresis has not been used for purification of low-abundance protein in complex sample matrix. Herein, lysozyme in complex egg white matrix was chosen as the model protein for demonstrating the purification of low-content peptide via an FFE coupled with gel fitration chromatography (GFC). The crude lysozyme in egg while was first separated via free-flow zone electrophoresis (FFZE). After that, the fractions with lysozyme activity were condensed via lyophilization. Thereafter, the condensed fractions were further purified via a GFC of Sephadex G50. In all of the experiments, a special poly(acrylamide- co-acrylic acid) (P(AM-co-AA)) gel electrophoresis and a mass spectrometry were used for identification of lysozyme. The conditions of FFZE were optimized as follows: 130 µL/min sample flow rate, 4.9 mL/min background buffer of 20 mM pH 5.5 Tris-Acetic acid, 350 V, and 14 °C as well as 2 mg/mL protein content of crude sample. It was found that the purified lysozyme had the purity of 80% and high activity as compared with its crude sample with only 1.4% content and undetectable activity. The recoveries in the first and second separative steps were 65% and 82%, respectively, and the total recovery was about 53.3%. The reasons of low recovery might be induced by diffusion of lysozyme out off P(AM-co-AA) gel and co-removing of high-abundance egg ovalbumin. All these results indicated FFE could be used as alternative tool for purification of target solute with low abundance.

Facile Counting of Ligands Capped on Nanoparticles via a Titration Chip of Moving Reaction Boundary Electrophoresis.

Absolute quantification of ligand capped on the surface of nanoparticles (NPs) has faced a great challenge without the use of complex inner standards (CIS). Herein, we proposed a facile electrophoresis titration (ET) model, designed an ET device, and developed a relevant method for counting the ligand on NPs without the use of CIS, based on moving reaction boundary (MRB). Furthermore, we conducted the relevant ET runs by using 3-mercaptopropionic acid (MPA) and quantum dots (QDs) as the model ligand and NPs, respectively. The experiments revealed that the ligand content of 1518 ± 295 obtained via an ET was close to the one of 1408 ± 117 determined via NMR, validating the ET model. Moreover, the experiments showed fair stability (RSD < 5.62%) and simplicity of ET without the use of CIS. Evidently, the ET model opens a window for facile assay of ligand capped on NPs.

Comparative proteomics analysis of microvesicles in human serum for the evaluation of osteoporosis.

Osteoporosis is an emerging health issue worldwide. Due to the decrease of bone mineral density and the deterioration of skeletal microarchitecture, osteoporosis could lead to increased bone fragility and higher fracture risk. Since lack of specific symptoms, novel serum proteomic indicators are urgently needed for the evaluation of osteoporosis. Microvesicles (MVs) are important messengers widely present in body fluids and have emerged as novel targets for the diagnosis of multiple diseases. In this study, MVs were successfully isolated from human serum and comprehensively characterized. Comparative proteomics analysis revealed differential MVs protein profiling in normal subjects, osteopenia patients, and osteoporosis patients. In total, about 200 proteins were identified and quantified from serum MVs, among which 19 proteins were upregulated (fold change >2) and five proteins were downregulated (fold change <0.5) in osteopenia group and osteoporosis group when compared with the normal group. Three protein candidates were selected for initial verification, including Vinculin, Filamin A, and Profilin 1. Profilin 1 was further pre-validated in an independent sample set, which could differentiate osteoporosis group from osteopenia group and normal group (p < 0.05). Our data collectively demonstrate that serum MVs proteome can be valuable indicators for the evaluation and diagnostics of bone loss disease.

A facile isoelectric focusing of myoglobin and hemoglobin used as markers for screening of chicken meat quality in China.

A novel analytical protocol was developed for general quality screening of chicken meat based on IEF and protein extraction. To demonstrate the developed protocol, 24 chickens were divided into three groups; each had eight chickens. The chickens in Group 1 were slaughtered by exsanguination, Group 2 asphyxiated in water, and that in Group 3 were infected by new castle disease virus. Proteins were extracted from the meat samples by using pure water as an extractant, separated by IEF, verified by western blot, and quantified via imaging analysis. The relevant experiments demonstrated that two myoglobin (Mb) bands were detected at pI 6.8 and 7.04 for all samples of Groups 1, 2, and 3, but there were additional hemoglobin (Hb) bands at pI 7.09 and 7.13 (P < 0.05) for the samples of Groups 2 and 3. The results implied that Hb bands might be a potential biomarker for the screening of chicken meat quality. The RSD values of two Mb bands (pI 6.8 and 7.04) in Group 1 were respectively 4.08 and 3.63%, the ones of two Hb bands (pI 7.09 and 7.13) in Group 2 were 3.66 and 2.10%, and those in Group 3 were 2.17% and 2.77%, respectively. All the RSD values indicated high stability and reliability of the developed protocol. Additionally, the protocol had a direct readout of protein bands in IEF without staining. However, it was time-consuming and had high cost. Even so, the relevant general method and finding have potential for screening of chicken meat quality.

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.

Narrow, Open, Tubular Column for Ultrahigh-Efficiency Liquid-Chromatographic Separation under Elution Pressure of Less than 50 bar.

We report that we can achieve extremely high separation efficiencies using a narrow, open, tubular (NOT) column for liquid-chromatographic separations, and we can carry out these separations under an elution pressure of no more than 50 bar. To improve the separation efficiency in packed-column liquid chromatography, one of the most effective approaches is to reduce the monodispersed-particle sizes. A direct consequence of reduced particle size is an increased elution pressure. High efficiencies have been obtained in ultrahigh-performance liquid chromatography (UPLC) using 1-2 µm or even submicron particles, and high elution pressures (greater than 1000 bar) are commonly used to carry out these separations. Open, tubular (OT) columns have been predicted to be the most efficient columns for high-efficiency liquid-chromatographic separations, as long as the column diameter is sufficiently small (1-2 µm). However, high efficiencies have not yet been publically reported, possibly because of the challenges (such as picoliter-volume detection, nanocapillary-column preparation, low sample loadability, etc.) of utilizing 1-2 µm diameter capillaries. In this paper, we show how we overcame these problems and achieved extremely high separation efficiencies using a 2 µm inner diameter capillary. We see 200+ apparent peaks with a peak capacity of 810 within 54 min when separating a sample from trypsin-digested cytochrome C, and we count 440 apparent peaks with a peak capacity of 1640 within 172 min when separating a sample from pepsin/trypsin-digested Escherichia coli cell lysate.

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.