Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence imaging.

The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03-10 µg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.

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.

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.

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.

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.

[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.

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.

[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.

A handheld contactless conductivity detector for monitoring the desalting of low-volume virus and cell samples.

Desalting of biosamples is crucial for analytical techniques intolerant to abundant salts. However, there is no simple tool to monitor the desalting of low-volume biosamples so far. Here we developed a handheld capacitively coupled contactless conductivity detector (hC4D) as a miniaturized device to measure the conductivity of 75 µL biosamples. Polyether-ether-ketone (PEEK) tubing was selected as the sample reservoir for sample loading via a pipette. Another pipetting of air pushed the sample solution out of the tubing to recollect the sample. Owing to the low sample consumption and easy sample recollection, hC4D is advantageous for testing expensive biosamples, such as viruses and cells. In addition, the whole process of sample injection, conductivity measurement, recollection, and calibration of conductivity can be completed within 1 min. To verify the feasibility of hC4D, we monitored the desalting progress of gel filtration (GF) of 200 µL blood samples, ultrafiltration (UF) of 300 µL virus samples, and dialysis of 7 mL cell samples. Three rounds of GF and UF completely removed the salts but led to poor sample recovery. In contrast, low concentrations of residual salts remained and better recovery was achieved after two rounds of GF and UF. We further utilized the hC4D to monitor the dialysis and tuned the salt concentration in the cell sample, such that we maintained the viability of cells in a low conductivity environment. These results indicated that hC4D is a promising tool for optimizing the desalting procedure of low-volume biosamples.

Point of care testing (POCT) of cholesterol in blood serum via a moving reaction boundary electrophoresis titration chip.

Cholesterol (CHO) in human blood is one of the most frequently and crucially quantified substances in diagnostic laboratories. However, visual and portable point of care testing (POCT) methods have been rarely developed for the bioassay of CHO in blood samples. Here, we developed an electrophoresis titration (ET) model, a chip device of ∼60 grams, and a quantification method for the POCT of CHO in blood serum based on a moving reaction boundary (MRB). In this model, the selective enzymatic reaction is integrated with an ET chip for visual and portable quantification. At first, CHO reacted with cholesterol oxidase (CHOx) in the anode well, producing H2O2 and cholest-4-en-3-one in the solution. H2O2 further oxidized the colorless and chargeless leucocrystal violet (LCV) dye into violet colored positively charged crystal violet (CV+) and, under the influence of the electric field, the CV+ migrates in the ET channels and is titrated by the alkali of sodium hydroxide immobilized in the ET channels. The length covered by the MRB was measured as a function of the CHO content. The relevant experiments validated the feasibility of the model and method. Furthermore, the experiments revealed the high selectivity, portability, and visuality of the ET-MRB model, device, and method. Finally, the experiments showed a fair sensitivity of LOD of 5 µM, good linearity of 10-1000 µM (r2 = 0.9919), fair stability (intra-day RSD of less than 5.09% and an inter-day RSD of less than 6.36%), and high recovery (99.4-105%). All the data and results indicate the potential of the ET-MRB model, chip device, and method for POCT of CHO in human blood samples.

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.

Isoelectric point barcode and similarity analysis with the earth mover's distance for identification of species origin of raw meat.

In this work, by combining the microcolumn isoelectric focusing (mIEF) and similarity analysis with the earth mover's distance (EMD) metric, we proposed the concept of isoelectric point (pI) barcode for the identification of species origin of raw meat. At first, we used the mIEF to analyze 14 meat species, including 8 species of livestock and 6 species of poultry, to generate 140 electropherograms of myoglobin/hemoglobin (Mb/Hb) markers. Secondly, we binarized the electropherograms and converted them into the pI barcodes that only showed the major Mb/Hb bands for the EMD analysis. Thirdly, we efficiently developed the barcode database of 14 meat species and successfully used the EMD method to identify 9 meat products thanks to the high throughput of mIEF and the simplified format of the barcode for similarity analysis. The developed method had the merits of facility, rapidity and low cost. The developed concept and method had evident potential to the facile identification of meat species.

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.

An efficient isoelectric focusing of microcolumn array chip for screening of adult Beta-Thalassemia.

Traditional capillary isoelectric focusing (cIEF), liquid chromatography (LC) and capillary zone electrophoresis (CZE) still suffered from low resolution for hemoglobinopathy screening. Herein, a 30-mm pH 5.2-7.8 microcolumn IEF (mIEF) array chip was developed for hemoglobinopathy screening. As a proof of concept, adult beta-thalassemia was chosen as a model disease. In the method, blood samples were hemolyzed via hemolysin solution and loaded into the microcolumn. The experiments showed that (i) the species of Hb A, F, A2 and variants were clearly separated in the chip, and the resolution was greatly higher than the ones of LC/CZE/cIEF; (ii) up to 24 samples could be simultaneously analyzed in 12-min run; (iii) the intraday and interday RSDs were respectively 3.32-4.91 % and 4.07-5.33 %. The assays of mIEF to total 634 samples were compared with the ones of LC (n = 327) and PCR (n = 307). The cutoff of 3.5 % HbA2 led to the sensitivity of 100 % and specificity of 89.1 % for the mIEF-based screening; and there was 96.7 % coincidence between the methods of mIEF and PCR if refer Hb A2 and F. The method had the merits of facility, efficiency, specificity and sensitivity in contrast to the currently-used methods, implying its potential to screening of beta-thalassemia and hemoglobinopathies.

In-Depth Metaproteomics Analysis of Oral Microbiome for Lung Cancer.

The human oral microbiome correlates with numerous diseases, including lung cancer. Identifying the functional changes by metaproteomics helps understand the disease-related dysbiosis, yet characterizing low-abundant bacteria is challenging. Here, we developed a free-flow isoelectric focusing electrophoresis-mass spectrometry- (FFIEF-MS-) based metaproteomics strategy to reduce host interferences and enrich low-abundant bacteria for in-depth interpretation of the oral microbiome. With our method, the number of interfering peptides decreased by 52.87%, whereas the bacterial peptides and species increased by 94.97% and 44.90%, respectively, compared to the conventional metaproteomics approach. We identified 3647 bacterial proteins, which is the most comprehensive oral metaproteomics study to date. Lung cancer-associated bacteria were validated among an independent cohort. The imbalanced Fusobacterium nucleatum and Prevotella histicola and their dysregulated functions in inhibiting immune response and maintaining cell redox homeostasis were revealed. The FFIEF-MS may serve as a valuable strategy to study the mechanisms between human diseases and microbiomes with broader applications.

Free-flow zone electrophoresis facilitated proteomics analysis of heterogeneous subpopulations in H1299 lung cancer cells.

Cancer cells are usually heterogeneous, whose subpopulations contain distinct biological information that related to their unique phenotype. However, preparative separating heterogeneous cells into subpopulations is very challenging, especially based on their surface charge status. Here, free-flow zone electrophoresis (FFZE) was employed to separate and fractionate lung cells. The experimental parameters for FFZE, including voltage, running speed, and buffer pH, were optimized and the optimized method was then implemented to separate MRC-5 and H1299 cells. Normal lung cells MRC-5 could be separated into one major peak and few minor peaks. For lung cancer cell line H1299, two major peaks and a few minor peaks were fractionated. Those prepared major peaks were subjected to quantitative proteomics analysis for heterogeneous subpopulation. For H1299 cells, 2076 proteins were identified in the first major peak, and 1398 proteins were revealed in the second major peak, which increased by 13% on average (n = 3) when compared to that of before FFZE fractionation. Furthermore, we found that H1299 cells in the first major peak have higher mobility than the cells in the second one. Many cell mobility and metastasis related proteins, such as RHBDF2, TRIM59, and ADAM17, were solely expressed in the first major peak of H1299 after FFZE, indicating our method can distinguish cancer cell subpopulations with higher metastatic phenotype. Our results showed that FFZE can effectively fractionate heterogeneous cell subpopulations, which might be used for the comprehensive analysis of unique phenotypes of different cancers.

Correction: Recent advances in microfluidic-based electroporation techniques for cell membranes.

Correction for 'Recent advances in microfluidic-based electroporation techniques for cell membranes' by Fei Wang et al., Lab Chip, 2022, 22, 2624-2646, https://doi.org/10.1039/D2LC00122E.

[In-site electrophoretic elution of excessive fluorescein isothiocyanate from fluorescent particles in gel for image analysis].

The sensitivity, accuracy, and efficiency of fluorescent particle detection can be improved by purifying the fluorescent-dye-labeled particles. In this study, an in-site model of electrophoretic elution (EE) was developed for the facile and efficient removal of unconjugated fluorescent dyes after labeling reactions, thereby facilitating the sensitive fluorescent imaging of proteins captured by microbeads. First, bovine serum albumin (BSA) and magnetic beads (MBs) were chosen as the model protein and particles, respectively, and an MBs-BSA complex was synthesized by mixing the beads with the BSA solution. Second, excessive fluorescein isothiocyanate (FITC) was added to the EP tube with MBs-BSA suspension for the fluorescent labeling of BSA, and a labeled compound was obtained after 8-h incubation in the dark at 4 ℃. The unpurified MBs-BSAFITC was obtained by removing the supernatant, leaving 5 µL of the residual solution in the EP tube. The obtained MBs-BSAFITC solution was added to a 50-µL phosphate buffer solution (PBST, containing 0.01% Triton X-100, pH 7.4). Third, gel suspension was prepared by mixing the MBs-BSAFITC solution with the low-gelling-temperature agarose gel (10 g/L) and filled into an electrophoresis channel. To demonstrate the high efficiency of the in-site model of EE for removing excessive FITC, a 10-mm hydrogel segment was prepared using MBs-BSAFITC sandwiched between two blank hydrogels and filled into a 50-mm-long electrophoresis tube (outer diameter: 5 mm; inner diameter: 3 mm) for the EE. Subsequently, the filled channel was set in an electrophoresis device to construct the in-site EE model. The particle size of the MBs was larger than the pore size of the gel, and the fluorescent beads were physically immobilized in the gel while the excessive FITC was removed from the channel by electrophoresis. Before an EE run, the original fluorescence image of the target gel was captured using a CCD camera. After the 30-min EE (50 V, 6 mA, pH 7.4 PBS), the fluorescence image was also recorded by the CCD camera. The fluorescent images were converted to a grayscale intensity map. To simplify the calculation, a simple fluorescent image analysis method was developed. The side view of the grayscale intensity map is a two-dimensional plot of peaks. Each peak indicates a fluorescent spot at a given position along the length of the channel when the distribution density of the particles is low, and the peak value is the grayscale intensity of the fluorescent spot. The statistical peak numbers and values can be used to approximate fluorescent spots on the image. After image processing and calculations, 27.8% of the average grayscale intensity of the fluorescent spot was retained, comparing the average gray value of the bright spot before and after EE, and 97.6% of excessive FITC in the channel was cleared, obtained by calculating the decreased background fluorescence grayscale intensity after EE. The particle-to-background signal ratio (P/B ratio, PBr) increased from 1.08 to 12.2 after EE with an exposure time of 1.35 s. In addition, different exposure times were explored during the fluorescence detection. Increasing the exposure time from 1.35 to 2.35 s enhanced PBr from 12.2 to 15.5, which could effectively increase the signal-to-noise ratio. An appropriate increase in exposure time also allowed the detection of many weak fluorescent particles that were previously undetectable, indicating increased sensitivity of the fluorescence detection. The EE model has the following advantages: (i) increase in specificity by eluting FITC absorbed to the surface of beads; (ii) high efficiency in the removal of free FITC with more than 97% clearance; (iii) rapid decrease in noise in the mass hydrogel (within 30 min). This method can be used in beads/spots-based immunoassay in gel, immuno-electrophoresis, and fluorescent staining of protein/nucleic acid bands in gel electrophoresis.

Recent advances in microfluidic-based electroporation techniques for cell membranes.

Electroporation is a fundamental technique for applications in biotechnology. To date, the ongoing research on cell membrane electroporation has explored its mechanism, principles and potential applications. Therefore, in this review, we first discuss the primary electroporation mechanism to help establish a clear framework. Within the context of its principles, several critical terms are highlighted to present a better understanding of the theory of aqueous pores. Different degrees of electroporation can be used in different applications. Thus, we discuss the electric factors (shock strength, shock duration, and shock frequency) responsible for the degree of electroporation. In addition, finding an effective electroporation detection method is of great significance to optimize electroporation experiments. Accordingly, we summarize several primary electroporation detection methods in the following sections. Finally, given the development of micro- and nano-technology has greatly promoted the innovation of microfluidic-based electroporation devices, we also present the recent advances in microfluidic-based electroporation devices. Also, the challenges and outlook of the electroporation technique for cell membrane electroporation are presented.