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Single-cell RNA sequencing (scRNA-seq) technologies are poised to reshape the current cell-type classification system. However, a transcriptome-based single-cell atlas has not been achieved for complex mammalian systems. Here, we developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices. Using Microwell-seq, we analyzed more than 400,000 single cells covering all of the major mouse organs and constructed a basic scheme for a mouse cell atlas (MCA). We reveal a single-cell hierarchy for many tissues that have not been well characterized previously. We built a web-based "single-cell MCA analysis" pipeline that accurately defines cell types based on single-cell digital expression. Our study demonstrates the wide applicability of the Microwell-seq technology and MCA resource.
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Análisis de Secuencia de ARN , Análisis de la Célula Individual , Células 3T3 , Animales , Costos y Análisis de Costo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Ratones , Especificidad de Órganos , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/economía , Análisis de la Célula Individual/economíaRESUMEN
Single-molecule spectroscopy offers state-resolved measurements on charge-transfer reactions of single semiconductor nanocrystals, leading to the discovery of up to six single-charge transfer reactions with seven transient states for single CdSe/CdS core/shell nanocrystals with water (or oxygen) as the hole (or electron) acceptors. Kinetic rates of three photoinduced single-hole transfer reactions decrease significantly upon increasing the number of excess electrons in a nanocrystal, mainly due to efficient Auger nonradiative recombination of the charged single excitons. Conversely, the kinetic rates of three single-electron transfer reactions of an unexcited nanocrystal increase proportionally to the number of excess electrons in it. Results here reveal that charge-transfer reactions of nanocrystals, at the center of nearly all their functions, could only be deciphered at a state-resolved level on a single nanocrystal. Size-dependent studies validate the weakly confined semiconductor nanocrystals, instead of strongly confined ones (quantum dots), as optimal candidates for photochemical and optoelectronic applications.
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With the increasing demand for trace sample analysis, injecting trace samples into liquid chromatography-mass spectrometry (LC-MS) systems with minimal loss has become a major challenge. Herein, we describe an in situ LC-MS analytical probe, the Falcon probe, which integrates multiple functions of high-pressure sample injection without sample loss, high-efficiency LC separation, and electrospray. The main body of the Falcon probe is made of stainless steel and fabricated by the computer numerical control (CNC) technique, which has ultrahigh mechanical strength. By coupling a nanoliter-scale droplet reactor made of polyether ether ketone (PEEK) material, the Falcon probe-based LC-MS system was capable of operating at mobile-phase pressures up to 800 bar, which is comparable to those of conventional ultraperformance liquid chromatography (UPLC) systems. Using the probe pressing microamount in situ (PPMI) injection approach, the Falcon probe-based LC-MS system showed high separation efficiency and good repeatability with relative standard deviations (RSDs) of retention time and peak area of 1.8% and 9.9%, respectively, in peptide mixture analysis (n = 6). We applied this system to the analysis of a trace amount of 200 pg of HeLa protein digest and successfully identified an average of 766 protein groups (n = 5). By combining in situ sample pretreatment at the nanoliter range, we further applied the present system in single-cell proteomic analysis, and 241 protein groups were identified in single 293 cells, which preliminarily demonstrated its potential in the analysis of trace amounts of samples with complex compositions.
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Presión , Humanos , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Nanotecnología , Polietilenglicoles/química , Péptidos/análisis , Cromatografía Líquida de Alta Presión , Células HeLa , Benzofenonas/análisis , Benzofenonas/química , Polímeros/química , Cetonas/química , Cetonas/análisis , Proteómica/métodosRESUMEN
Characterizing the profiles of proteome and metabolome at the single-cell level is of great significance in single-cell multiomic studies. Herein, we proposed a novel strategy called one-shot single-cell proteome and metabolome analysis (scPMA) to acquire the proteome and metabolome information in a single-cell individual in one injection of LC-MS/MS analysis. Based on the scPMA strategy, a total workflow was developed to achieve the single-cell capture, nanoliter-scale sample pretreatment, one-shot LC injection and separation of the enzyme-digested peptides and metabolites, and dual-zone MS/MS detection for proteome and metabolome profiling. Benefiting from the scPMA strategy, we realized dual-omic analysis of single tumor cells, including A549, HeLa, and HepG2 cells with 816, 578, and 293 protein groups and 72, 91, and 148 metabolites quantified on average. A single-cell perspective experiment for investigating the doxorubicin-induced antitumor effects in both the proteome and metabolome aspects was also performed.
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Proteoma , Espectrometría de Masas en Tándem , Humanos , Proteoma/metabolismo , Cromatografía Liquida , Metaboloma , Células HeLaRESUMEN
In this study, simple-structured wavelength sensors were developed by depositing two back-to-back Au/MAPbI3/Au photodetectors on an MAPbI3 single crystal. This sensor could quantitatively distinguish wavelengths. Further device analysis showed that both photodetectors possess entirely disparate optoelectronic properties. Consequently, the as-developed wavelength sensor could accurately distinguish incident-light wavelengths ranging from 265 to 860 nm with a resolution of less than 1.5 nm based on the relation between the photocurrent ratios of both photodetectors and the incident light wavelengths. Notably, a high resolution and wide detection range are among the optimum reported values for such sensors and enable full-color imaging. Furthermore, technology computer-aided design (TCAD) simulations showed that a mechanism involved in distinguishing wavelengths is attributed to the wavelength-dependent photon generation rate in MAPbI3 single crystals. The high-performance MAPbI3 wavelength sensor can potentially drive the research progress of perovskites in wavelength recognition and full-color imaging.
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Objective To analyze the current situation of dietary diversity and caregiver self-efficacy for complementary feeding among infants and young children aged 6 to 23 months in rural Nanchong city,Sichuan province,and to explore the relationship between dietary diversity and caregiver self-efficacy. Methods Multi-stage randomized cluster sampling method was used to select infants and young children aged 6 to 23 months and their caregivers in rural areas of Nanchong city,Sichuan province as the subjects.A structured questionnaire was designed to collect the basic information of the subjects,dietary diversity,and caregiver self-efficacy for complementary feeding.Multivariate Logistic regression was adopted to analyze the relationship between the dietary diversity and caregiver self-efficacy for complementary feeding of infants and young children. Results A total of 770 pairs of infants and young children and their caregivers were included.The minimum pass rate of dietary diversity was 61.56%(474/770) for all the infants and young children and 45.00%(108/240),69.16%(287/415),and 68.70%(79/115) for the infants and young children aged 6 to 11,12 to 17,and 18 to 23 months,respectively.The results of regression analysis showed that the caregiver self-efficacy of complementary feeding was a contributing factor for qualified dietary diversity of infants and young children in the case of other confounders being controlled(OR=1.42,95%CI=1.17-1.73,P<0.001). Conclusion The dietary diversity for infants and young children in rural Nanchong city,Sichuan province needs to be improved,and caregivers with higher self-efficacy of complementary feeding are more likely to provide diversified complementary feeding for infants and young children.
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Cuidadores , Autoeficacia , Niño , Lactante , Humanos , Preescolar , Dieta , ChinaRESUMEN
The continuing pursuit for a healthy life has led to the urgent need for on-site analysis. In response to the urgent needs of on-site analysis, we propose a novel concept, called lab at home (LAH), for building automated and integrated total analysis systems to perform chemical and biological testing at home. It represents an emerging research area with broad prospects that has not yet attracted sufficient attention. In this paper, we discuss the urgent need, challenges, and future prospects of this area, and the possible roadmap for achieving the goal of LAH has also been proposed.
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Age-related effects on motor asymmetry provide insight into changes in cortical activation during aging. To investigate potential changes in manual performance associated with aging, we conducted the Jamar hand function test and the Purdue Pegboard test on young and older adults. All tests indicated reduced motor asymmetry in the older group. Further analysis suggested that a significant decline in dominant (right) hand function resulted in less asymmetric performance in older adults. The finding is inconsistent with the application of the HAROLD model in the motor domain, which assumes improved performance in the non-dominant hand, leading to a reduction of motor asymmetry in older adults. Based on the manual performance in young and older adults, it is suggested that aging reduces manual asymmetry in both force production and manual dexterity due to the reduced performance of the dominant hand.
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Objective To investigate the status of exclusive breastfeeding and bottle feeding in remote rural areas of Sichuan province and explore the relationship between negative emotions of mothers and feeding patterns of infants.Methods Multistage cluster sampling was employed to select the infants aged 0-6 months and their mothers in remote rural areas of Sichuan province.A self-designed questionnaire was used to collect the demographic characteristics of mothers and infants and the basic family information.The Chinese version of Depression Anxiety Stress scale was used to evaluate mothers' negative emotions,and the Breastfeeding Self-efficacy scale to assess the confidence level of mothers' behavior of adhering to exclusive breastfeeding.Results Totally 723 pairs of infants and their mothers were included.The exclusive breastfeeding and bottle feeding rates were 34.16% (247/723) and 57.54% (416/723),respectively.Mothers with depression tendency were less likely to adopt exclusive breastfeeding (OR=0.532,95%CI=0.291-0.974,P=0.041) and more likely to adopt bottle feeding (OR=1.877,95%CI=1.054-3.344,P=0.033).Further subgroup analysis of breastfeeding self-efficacy showed that in the group of low self-efficacy,the mothers with depression tendency were less likely to adopt exclusive breastfeeding (OR=0.461,95%CI=0.236-0.902,P=0.024) and more likely to adopt bottle feeding (OR=1.968,95%CI=1.047-3.701,P=0.036) than the mothers without depression tendency.In the group of high self-efficacy,mothers' depression,anxiety,and stress tendency had no significant correlation with infant feeding patterns (all P>0.05).Conclusions The mothers in the remote rural areas of Sichuan province are more likely to employ bottle feeding than exclusive breastfeeding.The mothers with stronger depression tendency demonstrate lower possibility of exclusive breastfeeding and higher possibility of bottle feeding.Breastfeeding self-efficacy may affect the association between maternal depression and infant feeding patterns.
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Ansiedad , Depresión , Conducta Alimentaria , Madres , Humanos , Lactante , China , Autoeficacia , Pueblos del Este de Asia , Madres/psicología , Depresión/epidemiologíaRESUMEN
Objective To understand the current situation and explore the influencing factors of delay in seeking medical treatment for common symptoms of residents in the rural areas of Sichuan province. Methods In July 2019,multi-stage random sampling was carried out in Zigong city,Sichuan province,and the data were collected by face-to-face questionnaire interview.The residents who had lived at hometown for more than half a year in the past year and had seen a doctor in the most recent month were surveyed.Logistic regression was adopted to predict the influencing factors of delay in seeking medical treatment. Results A total of 342 subjects were enrolled,and the incidence of delay in seeking medical treatment was 13.45%(46/342).Compared with the young and middle-aged(<65 years)people,the elderly(≥65 years)people were more likely to have delay in seeking medical treatment (OR=2.187,95%CI=1.074-4.457,P=0.031).The rural residents who gave higher score of the overall quality of township health centers were less likely to have delay in seeking medical treatment (OR=0.854,95%CI=0.735-0.992,P=0.039). Conclusions The occurrence of delay in seeking medical treatment for common symptoms of rural residents in Sichuan province is low.Age and the overall quality evaluation of township health centers affect the occurrence of delay in medical treatment among the rural residents in Sichuan province.Efforts should be made to improve the awareness of disease prevention among the elderly in rural areas.The investment in health resources in township health centers should be increased to strengthen the introduction and training of talents.These measures can improve the health services in township health centers,guide residents to make timely use of health resources,and reduce the occurrence of delay in seeking medical treatment.
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Población Rural , Persona de Mediana Edad , Anciano , Humanos , Encuestas y Cuestionarios , Modelos Logísticos , China/epidemiologíaRESUMEN
OBJECTIVE: To investigate prenatal manifestations of Emanuel syndrome (ES) by retrospectively analyzing the results of prenatal diagnosis. METHODS: Thirteen fetuses were collected from five hospitals, of which six were confirmed with 47,der(22)t(11;22; ES) by karyotype and chromosomal microarray analysis (CMA). Seven were diagnosed with 46,t(11;22) balanced translocations by karyotype, including one de novo mosaic 46,XX,t(11;22). In 3/7, CMA was performed but did not identify chromosomal imbalances. The results of prenatal diagnoses were reviewed, including ultrasound examinations and genetic testing. RESULTS: In ES fetuses, the derivative 22 was consistently inherited from the mother, while in the balanced translocation group, the t(11;22) chromosome was of paternal origin in 3/6 cases, All ES fetuses presented with multiple abnormalities by ultrasound examinations. Diaphragm hernia (3/6), Dandy-Walker complex (3/6), and kidney aplasia (3/6), were the most common ultrasound findings. Sonographic soft markers such as increased nuchal translucency, increased nuchal fold thickness appeared in 3 cases and all of these were associated with other anomalies. However, none of the ultrasound findings differentiated ES from other genetic syndromes during fetal period. CONCLUSIONS: In this series, in fetuses with a der(22), the derivative chromosome was consistently of maternal origin. In contrast, 46,t(11;22) balanced translocations were of maternal or paternal origin. The results contribute to the literature regarding the fetal phenotype of ES. Due to the absence of specific features distinguishing ES from other genetic syndromes, confirming the diagnosis through invasive genetic testing is necessary.
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Medida de Translucencia Nucal , Diagnóstico Prenatal , Trastornos de los Cromosomas , Fisura del Paladar , Femenino , Feto/diagnóstico por imagen , Pruebas Genéticas/métodos , Cardiopatías Congénitas , Humanos , Discapacidad Intelectual , Hipotonía Muscular , Embarazo , Diagnóstico Prenatal/métodos , Estudios Retrospectivos , Translocación Genética , Ultrasonografía PrenatalRESUMEN
BACKGROUND: Acute kidney injury (AKI) is not a rare complication during anti-tuberculosis treatment in some patients with pulmonary tuberculosis (PTB). We aimed to develop a risk prediction model for early recognition of patients with PTB at high risk for AKI during anti-TB treatment. METHODS: This retrospective cohort study assessed the clinical baseline, and laboratory test data of 315 inpatients with active PTB who were screened for predictive factors from January 2019 to June 2020. The elements were analyzed by logistic regression analysis. A nomogram was established by the results of the logistic regression analysis. The prediction model discrimination and calibration were evaluated by the concordance index (C-index), ROC curve, and Hosmer-Lemeshow analysis. RESULTS: A total of 315 patients with PTB were enrolled (67 patients with AKI and 248 patients without AKI). Seven factors, including microalbuminuria, hematuria, cystatin-C (CYS-C), albumin (ALB), creatinine-based estimated glomerular filtration rates (eGFRs), body mass index (BMI), and CA-125 were acquired to develop the predictive model. According to the logistic regression, microalbuminuria (OR = 3.038, 95%CI 1.168-7.904), hematuria (OR = 3.656, 95%CI 1.325-10.083), CYS-C (OR = 4.416, 95%CI 2.296-8.491), and CA-125 (OR = 3.93, 95%CI 1.436-10.756) were risk parameter, while ALB (OR = 0.741, 95%CI 0.650-0.844) was protective parameter. The nomogram demonstrated good prediction in estimating AKI (C-index= 0.967, AUC = 0.967, 95%CI (0.941-0.984), sensitivity = 91.04%, specificity = 93.95%, Hosmer-Lemeshow analysis SD = 0.00054, and quantile of absolute error = 0.049). CONCLUSIONS: Microalbuminuria, hematuria, ALB reduction, elevated CYS-C, and CA-125 are predictive factors for the development of AKI in patients with PTB during anti-TB treatments. The predictive nomogram based on five predictive factors is achieved good risk prediction for AKI during anti-TB treatments.
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Lesión Renal Aguda , Tuberculosis Pulmonar , Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/epidemiología , Antituberculosos/efectos adversos , Creatinina , Humanos , Estudios Retrospectivos , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/tratamiento farmacológicoRESUMEN
Background/objective: Investigating the neural mechanisms underlying sport performance has been a research focus in the field of sport science. The current review aims to identify distinct characteristics between athletes and non-athletes at behavioral and neural levels. Further analysis was conducted as to potential reasons that contributed to the differences. Methods: Literature was searched through PubMed, ScienceDirect, Cochrane, EBSCO, and Web of Science for EEG studies that compared athletes with non-athletes or novices in behavioral performance and brain function. Results: The process of literature search and selection identified 16 studies that satisfied the predetermined inclusion criteria. Theta, alpha, and beta frequency bands were employed as the primary EEG measures of cortical activities in the included studies. Athletes indicated significant advantages over controls in behavioral performance, H e d g e s ' g = 0.42 , p = 0.02 , and brain function, H e d g e s ' g = 0.49 , p = 0.03 . Moderator analysis on behavioral performance indicated a large effect size in sport-related performance, H e d g e s ' g = 0.90 , p = 0.01 , but a small, non-significant effect size in general tasks, H e d g e s ' g = 0.14 , p = 0.44 . Conclusions: Superior performance in sport-related tasks mostly contributed to athletes' significant advantage in behavioral performance. Additionally, favorable profiles of brain function associated with athletes included neural efficiency, increased cortical asymmetry, greater cognitive flexibility, and precise timing of cortical activation. Applying EEG technique to sport has shown promising directions in performance improvement and talent identification for young athletes.
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Herein, we describe an in situ analysis probe, Petrel probe, highly integrating multiple functions of in situ sampling, in situ sample injection, high-performance liquid chromatography (HPLC) separation, and mass spectrometry (MS) electrospray. The Petrel probe was fabricated based on a single capillary, which consists of a micrometer-sized hole for sampling, a packed column for LC separation, and a tapered tip for MS electrospray. The design of the Petrel probe was optimized to obtain higher structural strength, and a polytetrafluoroethylene (PTFE) chip was used for sealing the probe-sampling hole to meet the high-pressure (â¼30 MPa) requirement of LC manifold. On the basis of the Petrel probe, we developed a novel valveless LC injection method, that is, the probe pressing microamount in situ (PPMI) injection method, which performs sample injection by pressing the probe-sampling hole on the PTFE chip, using the mobile phase to dissolve the sample dry spot in the sampling area on the chip, and injecting it into the LC column under high-pressure conditions for separation and subsequent MS analysis. The LC-MS system with the PPMI injection method exhibits rapid injection and separation speed, as well as minimum injection dead volume. It can yield a high separation efficiency comparable to those of conventional HPLC systems. The present system was optimized using standard peptide samples, and four peptides were separated within 11 min in a probe with an effective column length of 5 cm, achieving the highest theoretical plate number up to â¼5,500,000/m. The system was also applied in the separation of cytochrome C digest to demonstrate its separation ability for complex samples, and 21 peptides were detected in 8 min with an amino-acid coverage of 83%.
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Péptidos , Espectrometría de Masa por Ionización de Electrospray , Animales , Aves , Cromatografía Líquida de Alta Presión , Cromatografía LiquidaRESUMEN
BACKGROUND: The anti-M antibody can lead to hemolytic disease of the fetus and newborn (HDFN) and adverse fetal outcomes, especially in the Asian population. However, fetal erythropoiesis resulting from M alloimmunization needs further investigation. STUDY DESIGN AND METHODS: We analyzed erythropoiesis in eight fetuses with M alloimmunization and compared them with the fetuses affected by anti-D. They were matched as pairs according to the gestational age of diagnosis and the hematocrit before treatment. Paired t-tests or paired Wilcoxon rank-sum tests were conducted to compare the difference in the cord blood indexes. Pearson correlation analysis was used to evaluate the correlativity between hematocrit and the reticulocyte percentage in the two groups. RESULTS: The fetuses in the MN group had lower reticulocyte count and percentage than those in the RhD group (p < .05). All of the fetal reticulocyte production indexes (RPIs) in the MN group were less than 2, indicating an inadequate hemopoietic response to anemia, while the majority of the RPIs in the RhD group (85.7%) were significantly higher (p = .003), with 6 cases greater than 2.5. Hematocrit was negatively correlated with reticulocyte percentage (y = 54.7-171.7x, r2 = 0.825, p = .005) in the RhD group, while no significant correlation was found in the MN group. No difference in the number of IUT, interval, or the fetal outcome was found between the two groups. CONCLUSION: Fetal reticulocytopenia provided direct evidence of an inadequate hemopoietic response in HDFN due to anti-M, leading to hyporegenerative anemia. Once the IgG component of anti-M is detected, close monitoring should be considered.
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Anemia/inmunología , Eritroblastosis Fetal/inmunología , Feto/inmunología , Inmunoglobulina M/inmunología , Isoanticuerpos/inmunología , Adulto , Anemia/etiología , Anemia/fisiopatología , Anemia/terapia , Transfusión de Sangre Intrauterina , Eritroblastosis Fetal/fisiopatología , Eritroblastosis Fetal/terapia , Eritropoyesis , Femenino , Feto/fisiopatología , Humanos , Recién Nacido , Masculino , Embarazo , Reticulocitosis , Globulina Inmune rho(D)/inmunología , Resultado del Tratamiento , Adulto JovenRESUMEN
In situ sampling mass spectrometry (MS) systems can achieve rapid analysis of samples, while most of them do not have the pretreatment capability of chromatographic separation. This Article describes the design, fabrication, and application of a swan-shaped in situ sampling MS probe with liquid chromatography (LC) separation capacity. The LC-Swan probe was fabricated based on a single capillary with a micrometer-sized hole at its U-shaped bottom for sampling, a monolithic column for separation, and a tapered tip for electrospray. Four functions including in situ sampling, sample injection, chromatographic separation, and MS electrospray were integrated in the LC-Swan probe. Direct sampling and contacting-dissolution-injection sampling modes were developed to perform in situ sampling and injection of liquid samples and dry spot samples, respectively, in the high flow-resistance LC system. A pressing-sealing method was also developed using a polydimethylsiloxane (PDMS) sealer to achieve the sealing of the probe sampling hole during the high-pressure chromatographic separation process. The LC-Swan probe-based system exhibited effective desalting capacity in the analysis of angiotensin II with similar relative standard deviations (RSDs) of retention time and peak area below 3% and 19% (n = 3) for both salt-containing and salt-free samples. The present system was applied for analyzing cytochrome C digest to test its separation capability for samples with complex compositions, and 19 peptides were detected in 13 min with an amino acid coverage of 85%. We also applied the system in metabolite analysis of mouse organ sections of brain, liver, and kidney to preliminarily demonstrate its application potential in MS imaging analysis.
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Espectrometría de Masas/métodos , Sondas Moleculares/química , Péptidos/análisis , Animales , Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión , Citocromos c/metabolismo , Riñón/química , Riñón/metabolismo , Hígado/química , Hígado/metabolismo , Ratones , Péptidos/química , Propranolol/análisis , Propranolol/metabolismoRESUMEN
The further miniaturization of liquid-phase microextraction (LPME) systems has important significance and major challenges for microscale sample analysis. Herein, we developed a rapid and flexible droplet-droplet microfluidic microextraction approach to perform nanoliter-scale miniaturized sample pretreatment, by combining droplet-based microfluidics, robotic liquid handling, and LPME techniques. Differing from the previous microextraction methods, both the extractant and sample volumes were decreased from the microliter scale or even milliliter scale to the nanoliter scale. We utilized the ability of a liquid-handling robot to manipulate nanoliter-scale droplets and micrometer-scale positioning to overcome the scaling effect difficulties in performing liquid-liquid extraction of nanoliter-volume samples in microsystems. Two microextraction modes, droplet-in-droplet microfluidic microextraction and droplet-on-droplet microfluidic microextraction, were developed according to the different solubility properties of the extractants. Various factors affecting the microextraction process were investigated, including the extraction time, recovery method of the extractant droplet, static and dynamic extraction mode, and cross-contamination. To demonstrate the validity and adaptability of the pretreatment and analysis of droplet samples with complex matrices, the present microextraction system coupled with MALDI-TOF mass spectrometry (MS) detection was applied to the quantitative determination of 7-ethyl-10-hydroxylcamptothecin (SN-38), an active metabolite of the anticancer drug irinotecan, in 800-nL droplets containing HepG2 cells. A linear relationship (y = 0.0305x + 0.376, R2 = 0.984) was obtained in the range of 4-100 ng/mL, with the limits of detection and quantitation being 2.2 and 4.5 ng/mL for SN-38, respectively.
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The efficacy of targeted therapy is associated with multi-gene mutation status. Carrying out effective multi-genotyping analysis in combination has been a challenge in clinical settings. We therefore developed a droplet-based capillary electrophoresis (CE) system coupled with PCR-restriction fragment length polymorphism (PCR-RFLP) technology to detect multi-gene mutations from a small volume of samples. A 16 × 16 200-nL droplet array for sample encapsulation was constructed on a glass chip. The electrophoresis system consisted of a tapered vertical capillary filled with polyvinylpyrrolidone, a laser-induced fluorescence detector, and a high voltage power supply. Notably, a droplet-based electrokinetic sample introduction method and a "â©" shape capillary were developed to facilitate consecutive droplet sampling using a home-made automatic control module. The DL2000 DNA marker was consecutively separated, achieving high migration time and plate number reproducibility. The system was further applied to detect PCR-RFLP products. For colorectal cancer (CRC) cell lines, KRAS, BRAF, and PIK3CA were genotyped with a sensitivity of 0.25%. For CRC patient specimens, 30 samples were consecutively and automatically multi-genotyped without inter-sample contamination, with a lowest mutation frequency of 0.37%. For the first time, we developed a droplet-based CE system for consecutive DNA analysis with low sample consumption. This automated CE system could be further developed to integrate the full process of gene mutation detection, serving as a more effective platform for individualised therapy.
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Neoplasias Colorrectales/genética , Electroforesis Capilar/métodos , Mutación , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/genética , Neoplasias Colorrectales/patología , Genes ras , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Reproducibilidad de los ResultadosRESUMEN
The authors would like to call the reader's attention to the fact that unfortunately the name of Jianzhang Pan was missing as co-author of this contribution.