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 (<i>R</i>_MRB) 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 <i>R</i>_MRB value, was as a function of protein content. Thus, the <i>R</i>_MRB 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 <i>R</i>_MRB 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)

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 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

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

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