[Detection and analysis of moving reaction boundary-based electrophoresis distance using smartphone images].

Electrophoresis titration (ET) based on the moving reaction boundary (MRB) theory can detect the analyte contents in different samples by converting content signals into distance signals. However, this technique is only suitable for on-site qualitative testing, and accurate quantification relies on complex optical equipment and computers. Hence, applying this method to real-time point-of-care testing (POCT) is challenging. In this study, we developed a smartphone-based ET system based on a visual technique to achieve real-time quantitative detection. First, we developed a portable quantitative ET device that can connect to a smartphone; this device consisted of five components, namely, an ET chip, a power module, a microcontroller, a liquid crystal display screen, and a Bluetooth module. The device measured 10 cm×15 cm×2.5 cm, weighed 300 g, and was easy to hold. Thus, it is suitable for on-site testing with a run time of only 2-4 min. An assistant mobile software program was also developed to control the device and perform ET. The colored electrophoresis boundary can be captured using the smartphone camera, and quantitative detection results can be obtained in real time. Second, we proposed a quantitative algorithm based on ET channels. The software was used to recognize the boundary migration distance of three channels, a standard curve based on two given contents of the standards was established using the two-point method, and the content of the test sample was calculated. Human serum albumin (HSA) and uric acid (UA) were used as a model protein and biosample, respectively, to test the performance of the detection system. For HSA detection, different HSA solutions were mixed with a polyacrylamide gel (PAG) stock solution, phenolphthalein was added as an indicator, and sodium persulfate and tetramethyl ethylenediamine (TEMED) were used to promote polymerization to form a gel. For UA detection, agarose gel was filled into the ET channel, the UA sample, urate oxidase, and leucomalachite green were added into the anode cell and incubated for 20 min. ET was then performed. The fitting goodness (R2) values of HSA and UA were 0.9959 and 0.9935, respectively, with a linear range of 0.5-35.0 g/L and a log-linear range of 100-4000 µmol/L. The limits of detection for HSA and UA were 0.05 g/L and 50 µmol/L, respectively, and the corresponding relative standard deviations (RSDs) were not greater than 2.87% and 3.21%, respectively. These results demonstrate that the detection system has good accuracy and sensitivity. Clinical samples collected from healthy volunteers were used as target blood samples, and the developed system was used to measure serum total protein and UA levels. Serum samples from five volunteers were selected, standard curves of total serum protein and UA were established, and the test results were compared with hospital standard testing results. The relative errors for serum total protein and UA were less than 6.03% and 6.21%, respectively, and the corresponding RSDs were less than 3.72% and 5.84%, respectively. These findings verify the accuracy and reliability of the proposed detection system. The smartphone-based ET detection system introduced in this paper presents several advantages. First, it enables the portable real-time detection of total serum protein and UA. Second, compared with traditional ET strategies based on colored boundaries, it does not rely on optical detection equipment or computers to obtain quantitative detection results; as such, it can reduce the complexity of the operation and provide portability and real-time metrics. Third, the detection of two biomarkers, serum total protein and UA, is achieved on the same device, thereby improving the multitarget detection potential of the ET method. These advantages render the developed method a promising detection platform for clinical applications and real-time POCT.

[Determination of human serum total protein via electrophoresis titration and capacitively coupled contactless conductivity detection].

Serum total protein refers to the sum of all proteins in the serum, and its content determination is relevant to human health monitoring and disease diagnosis. However, existing detection techniques present a number of limitations; for example, the Kjeldahl method suffers from the negative effects of interfering substances such as non-protein nitrogen (NPN). Although the electrophoresis titration (ET) method has solved interference problems to some extent, the current ET technique relies on optical detection methods, which increases the tediousness of the operation. This study addresses the challenge of accurate serum total protein detection by combining the traditional ET technique with capacitively coupled contactless conductivity detection (C4D). The research contributions of this work are multifold. First, it presents the first development of an ET-C4D detection system, which consists of six components: an ET power module, an ET chip, a C4D sensing module, a detection module, a data acquisition card, and software. The developed system can capture the conductivity of substances in the channel using the software developed by our laboratory during ET. The detection system can be used to quantify the total protein content in human serum without the addition of specific labeling reagents or using optical detection equipment, and its running time is approximately 300 s. Second, this research proposes the corresponding principle of the system. Under an electric field, ion migration results in different pH levels before and after the boundary, leading to a protein surface charge difference. The maintenance of the electrical neutrality of the substances in the detection channel is related to the protein surface charge; therefore, the ion concentration distribution of the substances in the detection channel changes as the protein surface charge varies. A plot of conductivity as a function of running time showed an "inverted clock shape", first falling and then rising. Owing to the addition of different types and concentrations of proteins, the microenvironment of the entire system changes, resulting in different changes in conductivity. Third, the performance of the detection system was tested using human serum albumin (HSA) standard protein, which was mixed with polyacrylamide gel (PAG) mother liquor, riboflavin, etc., and irradiated under ultraviolet light for 10 min to form a gel. The ET experiments were then carried out. The shape of the conductivity curve was consistent with the proposed principle, and the higher the HSA concentration, the lower the conductivity curve trough, followed by a lagged time of the trough. Quantitative analysis of the conductivity signals showed that the linear range was 0.25-3.00 g/L, with a linearity of up to 0.98. The limit of detection (LOD) was 0.01 g/L, the relative standard deviation (RSD) was 1.90%, and the relative error of the test values was <7.20%, indicating the good detection stability and sensitivity of the system. Clinical samples collected from healthy volunteers were used as target blood samples for serum total protein content measurement using our detection system. Blood samples from a volunteer were used to obtain a standard curve, and the serum samples of other four volunteers were selected for ET-C4D and biuret detection. The results showed that the relative errors between the two methods were within 4.43%, indicating the accuracy and reliability of the detection system. The advantages of the ET-C4D detection system proposed in this paper are as follows: (i) ET-C4D realizes the rapid detection of total serum protein content based on the ET technique; (ii) compared with the traditional protein ET technique, the ET-C4D method does not rely on specific labeling components or optical detection equipment, thereby reducing the complexity of the operation; and (iii) the output signal of ET-C4D can be used for quantitative analysis with excellent analytical performance and high accuracy. These merits highlight the potential of the developed system for clinical application and biochemical analysis.

Discovery of small extracellular vesicle proteins from human serum for liver cirrhosis and liver cancer.

Hepatocellular carcinoma (HCC) is a common neoplastic transformation of the hepatocytes, which has high morbidity and mortality worldwide, particularly in Eastern Asia. HCC is also developed as a consequence of chronic liver cirrhosis, and both diseases are difficult to diagnosis and differentiate. Accurate noninvasive biomarkers for HCC and cirrhosis are urgently needed. In the search for novel candidates, small extracellular vesicles (sEVs) were isolated from the serum of liver cancer patients, liver cirrhosis patients, healthy control subjects, as well as the culture media of hepatocellular carcinoma cells (HepG2) and normal hepatocyte cells (Lo2). Isolated sEVs were confirmed by size distribution analysis, morphological analysis, and surface biomarker tests. Mass spectrometry based label-free quantification revealed 61 and 63 differentially expressed proteins in the serum sEVs of liver cirrhosis patients and liver cancer patients (p < 0.05), respectively. The proteomics data of cell-derived sEVs were combined for the selection of valuable candidates. Promising proteins were further verified by immunoassay, including thrombospondin-1 (THBS1), fibulin-1(FBLN1), and fibrinogen gamma chain (FGG), which could differentiate healthy control from liver cancer or liver cirrhosis. Our findings verified the hypothesis that cancer-related proteomics signatures are present in the sEVs of patient's serum and might be monitored for the evaluation of liver cancer and liver cirrhosis.

Facile, Rapid, and Low-Cost Electrophoresis Titration of Thrombin by Aptamer-Linked Magnetic Nanoparticles and a Redox Boundary Chip.

An aptamer-linked assay of a target biomarker (e.g., thrombin) is facing the challenges of long-term run, complex performance, and expensive instrument, unfitting clinical diagnosis in resource-limited areas. Herein, a facile chip electrophoresis titration (ET) model was proposed for rapid, portable, and low-cost assay of thrombin via aptamer-linked magnetic nanoparticles (MNPs), redox boundary (RB), and horseradish peroxidase (HRP). In the electrophoresis titration-redox boundary (ET-RB) model, thrombin was chosen as a model biomarker, which could be captured within 15 min by MNP-aptamer 1 and HRP-aptamer 2, forming a sandwich complex of (MNP-aptamer 1)-thrombin-(HRP-aptamer 2). After MNP separation and chromogenic reaction of 3,3',5,5'-tetramethylbenzidine (TMB) within 10 min, an ET-RB run could be completed within 5 min based on the reaction between a 3,3',5,5'-tetramethylbenzidine radical cation (TMB•+) and l-ascorbic acid in the ET channel. The systemic experiments based on the ET-RB method revealed that the sandwich complex could be formed and the thrombin content could be assayed via an ET-RB chip, demonstrating the developed model and method. In particular, the ET-RB method had the evident merits of simplicity, rapidity (less than 30 min), and low cost as well as portability and visuality, in contrast to the currently used thrombin assay. In addition, the developed method had high selectivity, sensitivity (limit of detection of 0.04 nM), and stability (intraday: 3.26%, interday: 6.07%) as well as good recovery (urine: 97-102%, serum: 94-103%). The developed model and method have potential to the development of a point-of-care testing assay in resource-constrained conditions.

Isoelectric focusing array with immobilized pH gradient and dynamic scanning imaging for diabetes diagnosis.

A traditional immobilized pH gradient (IPG) has a high stability for isoelectric focusing (IEF) but suffers from time-consuming rehydration, focusing and staining-imaging as well as complex performance. To address these issues, an IEF system with an array of 24 IPG columns (10 mm × 600 µm × 50 µm) and dynamic scanning imaging (DSI) was firstly designed for protein focusing. Moreover, two IPG columns (pH 4-9 and pH 6.7-7.7 of 10 mm in length) were firstly synthesized for IEF. A series of experiments were carried out based on the IEF array. In contrast to a traditional IPG IEF with more than 10 h rehydration, 5-14 h IEF and ca 10 h stain-imaging, the IEF array had the following merits: 25 min rehydration for sample loading, 4 min IEF, and 2 min dynamics scanning of 24 columns, well addressing the issues of traditional IEF. Furthermore, the IEF array had fair sensitivity (LOD of 60 ng), good recovery (95%), and high stability (1.02% RSD for intra-day and 2.16% for inter-day). Finally, the developed array was successfully used for separation and determination of HbA1c (a key biomarker for diabetes diagnosis) in blood samples. All these results indicated the applicability of the developed IEF array to diabetes diagnosis.

Electrophoresis Titration Model of a Moving Redox Boundary Chip for a Point-of-Care Test of an Enzyme-Linked Immunosorbent Assay.

Enzyme-linked immunosorbent assays (ELISAs) have been widely used in clinical examination, food safety, and environmental analyses. However, they still face a great challenge in designing a device for a point-of-care test (POCT) due to its bulk optical detector and complexity. Herein an electrophoresis titration (ET) model of a moving redox boundary (MRB) was proposed for constructing an ET-ELISA chip of a POCT just with sextuplet electrode pairs and laminated cells. The chip had an anodic well, middle well, and cathode well which were connected by microchannels. The ELISA process was conducted in the bottom of the middle well, where horseradish peroxidase (HRP) catalyzed 3,3',5,5'-tetra-methyl benzidine (TMB) as a blue TMB dimer with two positive charges. Under an electrical field of 29 V, the TMB dimer migrated into the titration channel and reacted with the ascorbic acid, creating an MRB. The MRB motion was a function of antigen content, indicating a visual distance-based assay. As a proof of concept, a C-reactive protein was chosen as a model antigen. The experiments systemically validated the ET-ELISA model and method. Particularly, the chip was smartphone-detected, traditional power supply free, and did not use sulfuric acid used in typical ELISA, making the ET-ELISA method extremely simple, portable, and safe. The ET-ELISA has great potential to visual and portable ELISA in clinical medicine, the environment, and food safety immunoassay.

iPhone-imaged and cell-powered electrophoresis titration chip for the alkaline phosphatase assay in serum by the moving reaction boundary.

As a vital enzyme, alkaline phosphatase (ALP) has great clinical significance in diagnoses of bone or liver cancer, bone metastases, rickets, and extrahepatic biliary obstruction. However, there is still no really portable chip for the ALP assay in blood. Herein, a simple electrophoresis titration (ET) model was developed for ALP detection via a moving reaction boundary (MRB). In the model, ALP catalyzed the dephosphorylation of a 4-methylumbelliferyl phosphate disodium salt (4-MUP) substrate in the cathode well to 4-methylumbelliferone ([4-MU]-) with a negative charge and blue fluorescence under UV excitation. After the catalysis, an electric field was used between the cathode and the anode. Under the electric field, [4-MU]- moved into the channel and neutralized the acidic Tris-HCl buffer, resulting in the quenching of [4-MU]- and creating a MRB. The ET system just had an ET chip, a lithium cell, a UV LED and an iPhone used as a recorder, having no traditional expensive power supply and fluorescence detector. The relevant method was developed, and a series of experiments were conducted via the ET chip. The experiments showed: (i) a MRB could be formed between the [4-MU]- base and the acidic buffer, and the MRB motion had a linear relationship with the ALP activity, validating the ET model; (ii) the ET run was not impacted by many interferences, implying good selectivity; and (iii) the ET chip could be used for portable detection within 10 min, implying an on-site and rapid analysis. In addition, the ET method had a relatively good sensitivity (0.1 U L-1), linearity (V = 0.033A + 3.87, R2 = 0.9980), stability (RSD 2.4-6.8%) and recoveries (101-105%). Finally, the ET method was successfully used for ALP assays in real serum samples. All the results implied that the developed method was simple, rapid and low-cost, and had potential for POCT clinical ALP assays.

Graphene Oxide-Facilitated Comprehensive Analysis of Cellular Nucleic Acid Binding Proteins for Lung Cancer.

Nucleic acid binding proteins (NABPs) mediate a broad range of essential cellular functions. However, it is very challenging to comprehensively extract whole cellular NABPs due to the lack of approaches with high efficiency. To this end, carbon nanomaterials, including graphene oxide (GO), carboxylated graphene (cG), and carboxylated carbon nanotube (cCNT), were utilized to extract cellular NABPs in this study through a new strategy. Our data demonstrated that GO, cG, and cCNT could extract nearly 100% cellular DNA in vitro. Conversely, their RNA extraction efficiencies were 60, 50, and 29%, respectively, partially explaining why GO has the highest NABPs yield compared to cG and cCNT. We further found that ionic bond mediated by cations between RNA and functional groups of nanomaterials facilitated RNA absorption on nanomaterials. About 2400 proteins were successfully identified from GO-enriched NABPs sample, and 88% of annotated NABPs were enriched at least 2 times compared to cell lysate, indicating the high selectivity of our strategy. The developed method was further applied to compare the NABPs in two lung cancer cell lines with different tumor progression abilities. According to label-free quantification results, 118 differentially expressed NABPs were discovered and 6 candidate NABPs, including ACAA2, GTF2I, VIM, SAMHD1, LYAR, and IGF2BP1, were successfully validated by immunoassay. The level of SAMHD1 in the serum of lung cancer patients was measured, which significantly increased upon cancer progression. Our results collectively demonstrated that GO is an ideal nanomaterial for NABPs selective extraction, which could be broadly used in varied physiological and pathophysiological settings.

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.

Graphene and graphene oxide as a solid matrix for extraction of membrane and membrane-associated proteins.

The extraction of membrane proteins remain a challenge due to innate hydrophobicity, dynamic discrepancy, and restrain effect of membrane lipids. Nanomaterials with high surface area have competency of hydrophobic-hydrophobic lipid interactions. It is shown here that both graphene and graphene oxide dissolved in solubilization buffer are viable sorbents for efficient extraction of membrane proteins. LC-MS/MS analysis further revealed that graphene (50-200 nm) and graphene oxide (50-200 nm) can enrich more kinds of membrane proteins than a commercially available kit. Graphene was further applied to the enrichment of membrane proteins of normal cells as well as cancer cells, and 1079 and 872 proteins were identified, respectively, among which 56.5% and 60.5% were membrane proteins. In particular, 241 proteins were significantly regulated in cancer cells. Gene expression of 15 proteins was verified by qRT-PCR, and 4 of them were further quantified by immunoassay. These data collectively demonstrate that graphene has great potential to improve membrane protein extractions and thus can serve downstream cancer proteomics. Graphical abstract Two dimensional carbon nanomaterials, including graphene and graphene oxide, were employed as solid matrix to avoid lipid bilayer interference and enhance the extraction efficiency of membrane and membrane associated proteins. The strategy will benefit downstream membrane proteomics analysis.

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.

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.

Preparation of intact mitochondria using free-flow isoelectric focusing with post-pH gradient sample injection for morphological, functional and proteomics studies.

Mitochondria play essential roles in both energy metabolism and cell signaling, which are critical for cell survival. Although significant efforts have been invested in understanding mitochondrial biology, methods for intact mitochondria preparation are technically challenging and remain to be improved. New methods for heterogeneous mitochondria purification will therefore boost our understanding on their physiological and biophysical properties. Herein, we developed a novel recycling free-flow isoelectric focusing (RFFIEF) with post-pH gradient sample injection (post-PGSI) for preparative separation of mitochondria. Crude mitochondria of rabbit liver obtained from differential centrifugation were purified by the developed method according to their pI values as six fractions. Transmission electron microscope images revealed that intact mitochondria existed in two fractions of pH 6.24 and 6.61, degenerative mitochondria were in two fractions of pH 5.46 and 5.72, and inner membrane vesicles (IMVs) appeared in the fractions of pH 4.70 and 5.04. Membrane potential measurement proved a dramatic difference between intact mitochondria and IMVs, which reflected the bioactivity of obtained populations. Particularly, proteomics analyses revealed that more number of proteins were identified in the intact fractions than that of IMVs or crude mitochondria, which demonstrated that RFFIEF could be powerful tool for the preparation of intact organelle as well as their proteomic and in-depth biological analysis.

Systematic comparison of exosomal proteomes from human saliva and serum for the detection of lung cancer.

Circulating tumor exosomes harbor plenty of cancer biological information, which have emerged as promising targets for cancer early detection and diagnosis. Human serum and saliva are unique diagnostic body fluids, which contain numerous circulating exosomes. It is necessary to establish standardized isolation method and compare their proteome profiling for translational medicine. High abundant proteins in these body fluids were removed before exosomes isolation and obtained exosomes were further confirmed by morphology analysis and surface biomarker test. Label free quantification was applied to systematically compare the protein profiling in saliva and serum exosomes. 319 and 994 exosomal proteins were identified from saliva and serum by LC-MS/MS, respectively. To explore their utility for cancer proteomics, we systematically compared the proteome of saliva and serum exosomes from healthy subjects and lung cancer patients. In particular, 11 potential candidates were coincidently discovered in both body fluids of lung cancer patients. Our finding enforced the hypothesis that cancer related proteins were presented in saliva and serum exosomes, which promoted the unique features of exosomes in our body fluids. A circulating exosomes based body fluid test could be easily established for monitoring cancer once these candidates were validated.

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.

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.

Facile preparation of salivary extracellular vesicles for cancer proteomics.

Extracellular vesicles (EVs) are membrane surrounded structures released by cells, which have been increasingly recognized as mediators of intercellular communication. Recent reports indicate that EVs participate in important biological processes and could serve as potential source for cancer biomarkers. As an attractive EVs source with merit of non-invasiveness, human saliva is a unique medium for clinical diagnostics. Thus, we proposed a facile approach to prepare salivary extracellular vesicles (SEVs). Affinity chromatography column combined with filter system (ACCF) was developed to efficiently remove the high abundant proteins and viscous interferences of saliva. Protein profiling in the SEVs obtained by this strategy was compared with conventional centrifugation method, which demonstrated that about 70% more SEVs proteins could be revealed. To explore its utility for cancer proteomics, we analyzed the proteome of SEVs in lung cancer patients and normal controls. Shotgun proteomic analysis illustrated that 113 and 95 proteins have been identified in cancer group and control group, respectively. Among those 63 proteins that have been consistently discovered only in cancer group, 12 proteins are lung cancer related. Our results demonstrated that SEVs prepared through the developed strategy are valuable samples for proteomics and could serve as a promising liquid biopsy for cancer.

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.

Differential Proteomic Analysis of Human Saliva using Tandem Mass Tags Quantification for Gastric Cancer Detection.

Novel biomarkers and non-invasive diagnostic methods are urgently needed for the screening of gastric cancer to reduce its high mortality. We employed quantitative proteomics approach to develop discriminatory biomarker signatures from human saliva for the detection of gastric cancer. Salivary proteins were analyzed and compared between gastric cancer patients and matched control subjects by using tandem mass tags (TMT) technology. More than 500 proteins were identified with quantification, and 48 of them showed significant difference expression (p < 0.05) between normal controls and gastric cancer patients, including 7 up-regulated proteins and 41 down-regulated proteins. Five proteins were selected for initial verification by ELISA and three were successfully verified, namely cystatin B (CSTB), triosephosphate isomerase (TPI1), and deleted in malignant brain tumors 1 protein (DMBT1). All three proteins could differentiate gastric cancer patients from normal control subjects, dramatically (p < 0.05). The combination of these three biomarkers could reach 85% sensitivity and 80% specificity for the detection of gastric cancer with accuracy of 0.93. This study provides the proof of concept of salivary biomarkers for the non-invasive detection of gastric cancer. It is highly encouraging to turn these biomarkers into an applicable clinical test after large scale validation.