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.

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.

[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.

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.

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 protocol of rapid laboratory evolution by genome shuffling in Kluyveromyces marxianus.

Genome shuffling is a process to combine advantage traits by the recombination of the entire genome and it has been successfully applied in the laboratory evolution of various industrial microorganisms. However, genome shuffling has not been described in Kluyveromyces marxianus (KM), a promising yeast host for the expression of heterologous proteins. In this protocol, genome shuffling in KM is performed by sexual reproduction and is combined with high-throughput screening to obtain high-yielding strains. Notably, the screening of diploid clones risen from one mating mixture is carried out to improve the effectiveness of evolution. Mating-sporulation-mating cycles are repeated to obtain KM strain with ideal traits. •The method combines genome shuffling with high-throughput to achieve strains displaying high yielding of heterologous proteins.•This method can be applied to the genome shuffling of other species when only a few starting strains are available for sexual reproduction.

Improving the expression of a heterologous protein by genome shuffling in Kluyveromyces marxianus.

Genome shuffling is an efficient way to pool advantageous traits during positive selections of industrial microorganisms. In this study, for the first time, the effectiveness of genome shuffling to improve yielding of heterologous proteins was investigated in Kluyveromyces marxianus (KM), a promising yeast host. After two rounds of mating and screening, a novel KM strain, D2-13, was obtained which displayed a 5-fold increase of expression level of a heterologous protein comparing to its parental strains. A range of alleles linked with improved yielding were well preserved from a parental strain T1/E to D2-13, including one mutant allele of MTC6 known to attenuate autophagy. The results reflected an efficient pooling of advantageous alleles in our screen. Transcriptional analysis of D2-13, revealed that mRNA levels of genes implicated in protein folding, including those of AHA1, DNAJB13, and YGR250C, were significantly elevated, suggesting potential roles of these genes in promoting the expression of heterologous proteins. Our study not only indicates the applicability of genome shuffling in the optimization of KM host strains but also provided valuable clues to clarify the mechanisms underlying the high yielding of heterologous proteins.

Engineering Yarrowia lipolytica to Simultaneously Produce Lipase and Single Cell Protein from Agro-industrial Wastes for Feed.

Lipases are scarcely exploited as feed enzymes in hydrolysis of lipids for increasing energy supply and improving nutrient use efficiency. In this work, we performed homologous overexpression, in vitro characterization and in vivo assessment of a lipase from the yeast Yarrowia lipolytica for feed purpose. Simultaneously, a large amount of yeast cell biomass was produced, for use as single cell protein, a potential protein-rich feed resource. Three kinds of low cost agro-industrial wastes were tested as substrates for simultaneous production of lipase and single cell protein (SCP) as feed additives: sugarcane molasses, waste cooking oil and crude glycerol from biodiesel production. Sugarcane molasses appeared as the most effective cheap medium, allowing production of 16420 U/ml of lipase and 151.2 g/L of single cell protein at 10 liter fermentation scale. In vitro characterization by mimicking a gastro-intestinal environment and determination of essential amino acids of the SCP, and in vivo oral feeding test on fish all revealed that lipase, SCP and their combination were excellent feed additives. Such simultaneous production of this lipase and SCP could address two main concerns of feed industry, poor utilization of lipid and shortage of protein resource at the same time.