Clinical effectiveness of spindle-view intracytoplasmic sperm injection compared to conventional intracytoplasmic sperm injection in patients with poor ovarian response and previous implantation failure.

OBJECTIVE: This study aimed to determine the clinical advantage of spindle-view intracytoplasmic sperm injection (SVICSI; a novel technology) over conventional intracytoplasmic sperm injection (cICSI) in patients with poor ovarian response (POR) and previous implantation failure. METHODS: The study included 37 patients who underwent SVICSI followed by fresh embryo transfer (FET) at a single fertility clinic from January to December 2022, 58 patients who underwent cICSI followed by FET at the same fertility clinic from January to December 2021 as a control group. All study participants met the Bologna criteria for POR and had at least three or more previous failed embryo transfers. RESULTS: The number of blastocyst transfers was significantly higher in the SVICSI group than in the cICSI group. A good-quality cleavage embryo rate, blastocyst rate, and good-quality blastocyst rate were also significantly higher in the SVICSI group than in the cICSI group. There were no significant differences in the rates of fertilization, implantation, clinical pregnancy, or clinical abortion between the two groups. CONCLUSION: In patients with POR, those who underwent SVICSI appeared to have better embryos than those who underwent cICSI. However, whether SVICSI improved clinical outcomes such as implantation and pregnancy rates cannot be proven.

Relationship between hematologic parameters related to systemic inflammation and insulin resistance-associated metabolic parameters in women with polycystic ovary syndrome.

OBJECTIVE: The aim of the present study was to evaluate the associations between hematologic parameters related to systemic inflammation and insulin resistance-associated metabolic parameters in women with polycystic ovary syndrome (PCOS). METHODS: Eighty-two women between the ages of 18 and 35 years who were diagnosed with PCOS were included in this study. A 2-hour 75-g oral glucose tolerance test (OGTT) was administered to all study participants; fasting and postprandial glucose and insulin levels were measured simultaneously during the 2-hour OGTT. Hematologic parameters were derived from a standard complete blood count and a differential count of fasting-state blood samples. The correlations between hematologic parameters and insulin resistance-associated clinical and metabolic parameters were evaluated using the Spearman rank correlation and partial correlation coefficients. Hematologic parameters related to systemic inflammation were compared between the two groups, categorized by the presence or absence of insulin resistance. RESULTS: Significant differences in the absolute neutrophil count, absolute monocyte count, platelet count, and neutrophil-lymphocyte ratio were found between the insulin-resistant group and insulin-nonresistant group. Correlation analysis found that all hematological parameters, except for the platelet-lymphocyte ratio, were associated with at least one insulin resistance-associated metabolic parameter. However, these significant correlations between hematological and metabolic parameters were attenuated after controlling for the effects of other covariates using partial correlation analysis. CONCLUSION: The association between hematologic parameters indicative of systemic inflammation and insulin resistance-associated metabolic parameters seems to be strongly influenced by other anthropometric covariates in women with PCOS.

Do Second Graders Adjust Their Language by Discourse Context?

PURPOSE: Children's ability to adjust one's language according to discourse context is important for success in academic settings. This study examined whether second graders vary in linguistic and discourse features depending on discourse contexts, that is, when describing pictures in contextualized (describing the picture to an examiner while looking at it together) and decontextualized (pretending to describe the picture to a friend while sitting in front of the examiner) conditions. METHOD: A total of 330 English-speaking second graders in the United States (M age = 7.33 years; 53% boys; 55% Caucasian children, 35% African American children) described three pictures in contextualized and decontextualized conditions. Children's picture descriptions were transcribed verbatim and coded for linguistic (e.g., elaborated noun phrase) and discourse (e.g., proper character introduction, degree of decontextualization) features. RESULTS: Type-token ratio was higher in the contextualized condition than in the decontextualized condition, whereas certain types of elaborated noun phrases (e.g., simple descriptive noun phrase, noun phrase with postmodification), coordinating conjunctions, and nonclauses occurred more frequently in the decontextualized condition, controlling for total productivity and student demographics. The proportion of proper character introduction was higher in the decontextualized condition, whereas higher degrees of decontextualization and complex perspective-taking were found in the contextualized condition. CONCLUSION: Various linguistic and discourse cues illustrated the extent to which primary grade students employ their discourse knowledge when producing oral language.

Biosynthesis and applications of iron oxide nanocomposites synthesized by recombinant Escherichia coli.

Recombinant Escherichia coli (E. coli) strain that produces phytochelatin (PC) and/or metallothionein (MT) can synthesize various metal nanoparticles (NPs) by reducing metal ions. Here we report in vivo biosynthesis of iron oxide nanocomposites (NCs) using recombinant E. coli. We designed a strategy of biosynthesizing iron oxide NCs by first internalizing chemically synthesized iron oxide NPs, followed by the reduction of added metal ions on the surface of internalized NPs by PC and/or MT in E. coli. For this, chemically synthesized Fe3O4 NPs were internalized by recombinant E. coli, and then, Au and Ag ions were added for the biosynthesis of AuFe3O4 and AgFe3O4 NCs, respectively. The NCs synthesized were analyzed by transmission electron microscopy, UV-vis spectrophotometry, and X-ray diffractometry to characterize their shape, optical property, and crystallinity. The Fe3O4 NPs in the biosynthesized NCs allowed easy purification of the biosynthesized NCs by applying a magnetic field. The AuFe3O4 NCs were used for enzyme-linked immunosorbent assay to detect prostate-specific antigen protein, while AgFe3O4 NCs were utilized for the antimicrobial application with low minimum inhibitory concentration. As recombinant E. coli can uptake and reduce various NPs and metal ions, biosynthesis of a wide range of NCs as new nanomaterials will be possible for diverse applications. KEY POINTS: • AuFe3O4 and AgFe3O4 nanocomposites were synthesized by recombinant E. coli. • Escherichia coli synthesized different iron oxide NCs depending on the metal ions to be added. • Biosynthesized AuFe3O4 NC was used for ELISA and AgFe3O4 NC for antimicrobial tests.

Modular 3D In Vitro Artery-Mimicking Multichannel System for Recapitulating Vascular Stenosis and Inflammation.

Inflammation and the immune response in atherosclerosis are complex processes involving local hemodynamics, the interaction of dysfunctional cells, and various pathological environments. Here, a modular multichannel system that mimics the human artery to demonstrate stenosis and inflammation and to study physical and chemical effects on biomimetic artery models is presented. Smooth muscle cells and endothelial cells were cocultured in the wrinkled surface in vivo-like circular channels to recapitulate the artery. An artery-mimicking multichannel module comprised four channels for the fabrication of coculture models and assigned various conditions for analysis to each model simultaneously. The manipulation became reproducible and stable through modularization, and each module could be replaced according to analytical purposes. A chamber module for culture was replaced with a microfluidic concentration gradient generator (CGG) module to achieve the cellular state of inflamed lesions by providing tumor necrosis factor (TNF)-α, in addition to the stenosis structure by tuning the channel geometry. Different TNF-α doses were administered in each channel by the CGG module to create functional inflammation models under various conditions. Through the tunable channel geometry and the microfluidic interfacing, this system has the potential to be used for further comprehensive research on vascular diseases such as atherosclerosis and thrombosis.

Preclinical assessment of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells for neonatal hypoxic-ischaemic brain injury.

Hypoxic-ischaemic encephalopathy (HIE) is a type of brain injury affecting approximately 1 million newborn babies per year worldwide, the only treatment for which is therapeutic hypothermia. Thrombin-preconditioned mesenchymal stem cells (MSCs) exert neuroprotective effects by enriching cargo contents and boosting exosome biogenesis, thus showing promise as a new therapeutic strategy for HIE. This study was conducted to evaluate the tissue distribution and potential toxicity of thrombin-preconditioned human Wharton's jelly-derived mesenchymal stem cells (th-hWJMSCs) in animal models before the initiation of clinical trials. We investigated the biodistribution, tumorigenicity and general toxicity of th-hWJMSCs. MSCs were administered the maximum feasible dose (1 × 105 cells/10 µL/head) once, or at lower doses into the cerebral ventricle. To support the clinical use of th-hWJMSCs for treating brain injury, preclinical safety studies were conducted in newborn Sprague-Dawley rats and BALB/c nude mice. In addition, growth parameters were evaluated to assess the impact of th-hWJMSCs on the growth of newborn babies. Our results suggest that th-hWJMSCs are non-toxic and non-tumorigenic in rodent models, survive for up to 7 days in the brain and hold potential for HIE therapy.

Does Perspective Taking Matter for Writing? Perspective Taking in Source-Based Analytical Writing of Secondary Students.

Perspective taking, one's knowledge of their own mental and emotional states and inferences about others' mental and emotional states, is an important skill for writing development. In the present study, we examined how perspective taking is expressed in writing and how it is related to overall writing quality. We analyzed seventh graders' source-based analytical essays (N = 195) to investigate (1) the extent to which students incorporated perspective taking in their essays, (2) how the extent of perspective taking in essays differ by students' sex and English learner status, and (3) the extent to which perspective taking in writing is associated with overall writing quality. Findings revealed that students wrote more from their own perspective than that of others. Moreover, the results of multi-level analyses suggested that female students exhibited more varied perspectives but there was no meaningful difference by English learner status. Lastly, greater extent of perspective taking, particularly that of higher level of perspectives (i.e., dual perspective), was associated with better writing quality, after accounting for students' demographic backgrounds (e.g., sex, poverty status, English learner status) and essay length. These results underscore the importance of writing from multiple perspectives on writing quality.

Biomarker barcodes: multiplexed microfluidic immunohistochemistry enables high-throughput analysis of tissue microarray.

We present a multiplexed microfluidic immunohistochemistry (IHC) technology that enables high-throughput analysis of tissue microarrays (TMAs) using the patterns of biomarker barcodes, which consist of a series of expressed linear patterns of specific biomarkers. A multichannel poly(dimethylsiloxane) microfluidic device was reversibly assembled by the pressure of simple equipment for multiplexed IHC on each core of TMA or cell microarray (CMA) section slides. By injecting primary antibodies from different biomarkers independently into each channel, multiplexed immunostaining can be performed on each core of TMA. We confirmed the equal immunostaining quality regardless of the channel orders and core positions in the slide. Four different biomarkers (ER, PR, HER2, and Ki67) were used for the demonstration of distinctive expression patterns on CMAs which consist of six different breast cancer cell lines, and it was confirmed that these bar-like signals could be a biomarker barcode for the TMA core. A biomarker barcode of breast cancer patient-derived TMA was quickly scanned by a slide scanner and compared to the conventional method for breast cancer diagnosis. This "barcode-IHC" concept, which has been verified by performing multiplexed microfluidic IHC on CMA and TMA samples, provides high reproducibility and the potential of high-throughput screening with molecular diagnostic capability.

Theory of mind, mental state talk, and discourse comprehension: Theory of mind process is more important for narrative comprehension than for informational text comprehension.

We investigated the relations among theory of mind (ToM), mental state talk, and discourse comprehension. Specifically, we examined the frequency of mental state talk in children's oral recall of narrative texts and informational texts as well as relations among ToM, mental state talk (inclusion of mental state words in the recall of narrative and informational texts), and narrative and informational text comprehension. Results from children in Grade 4 (N = 132; Mage = 10.39 years) revealed that a greater number of mental state talk instances appeared in children's recall of narrative texts than in their recall of informational texts, but the mean number also differed across texts within a genre. ToM skill predicted the extent of mental state talk in narrative texts and informational texts, and the relation was stronger for narrative texts than for informational texts, after accounting for vocabulary, grammatical knowledge, working memory, and attentional control. Mental state talk in narrative texts was extremely strongly related to narrative comprehension, whereas mental state talk in informational texts was weakly related to informational text comprehension. Results suggest that ToM skill relates to mental state talk in the recall of texts, and both ToM and mental state talk play greater roles in comprehension of narrative texts than in comprehension of informational texts.

Safety evaluation of fermented Platycodon grandiflorus (Jacq.) A.DC. extract: Genotoxicity, acute toxicity, and 13-week subchronic toxicity study in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Platycodon grandiflorus (Jacq.) A.DC. is a well-known traditional herbal medicine administered for bronchitis and inflammatory diseases. Especially, anti-inflammatory effect of fermented P. grandiflorus (Jacq.) A.DC. extract (FPGE) was higher than that of P. grandiflorus (Jacq.) A.DC. extract. However, toxicological information for FPGE is lacking. AIM OF THE STUDY: In this study, we establish a toxicological profile for FPGE by testing genotoxicity, acute and 13-week subchronic toxicity. MATERIALS AND METHODS: FPGE was evaluated with bacterial reverse mutation, chromosome aberration, and micronucleus test. For the acute- and 13-week subchronic toxicity tests, FPGE was administered orally at doses of 0, 750, 1500, and 3000 mg/kg in SD rats. RESULTS: The results of the genotoxic assays indicated that FPGE induced neither mutagenicity nor clastogenicity. The acute toxicity test showed that FPGE did not affect animal mortality, clinical signs, body weight changes, or microscopic findings at ≤ 3000 mg/kg. The approximate lethal dose (ALD) of FPGE in SD rats was >3000 mg/kg. For the 13-week subchronic toxicity assay, no FPGE dose induced any significant change in mortality, clinical signs, body or organ weight, food consumption, ophthalmology, urinalysis, hematology, serum chemistry, gross findings and histopathologic examination in either SD rat sex. The rat no observed adverse effects level (NOAEL) for FPGE was set to 3000 mg/kg. CONCLUSIONS: The present study empirically demonstrated that FPGE has a safe preclinical profile and indicated that it could be safely integrated into health products for atopic dermatitis treatment.

2,3,7,8-Tetrachlorodibenzo-p-dioxin prompted differentiation to CD4+CD8-CD25+ and CD4+CD8+CD25+ Tregs and altered expression of immune-related genes in the thymus of chicken embryos.

The chicken (Gallus gallus), which has three aryl hydrocarbon receptor (AHR) isoforms (ckAHR1, ckAHR2, and ckAHR1ß) and two AHR nuclear translocator (ARNT) isoforms (ckARNT1 and ckARNT2), is highly sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and can serve as an avian model to gain an understanding of the mechanism underlying dioxin toxicity. To elucidate the mechanism of TCDD-induced immunotoxicity in avian species, we treated chicken embryos in ovo with graded concentrations of TCDD (1.5, 2.5, 3.0, 3.3, 3.5, and 4.0 µM). Initially, we measured mRNA expression levels of ckAHR and ckARNT isoforms and analyzed the T cell populations and transcriptome in the thymuses of TCDD-treated chicken embryos. Quantitative polymerase chain reaction analysis revealed that mRNA expressions of ckAHR1 and ckARNT2 were dominant in the thymus. Severe weight loss and thymus atrophy were observed in the TCDD-treated embryos. Immunophenotyping analyses demonstrated significant increases in CD4+CD8-CD25+ and CD4+CD8+CD25+ regulatory T cells (Tregs) populations following TCDD exposure, suggesting that TCDD suppresses T cell-mediated immune responses in chicken embryos. In addition, thymic transcriptome analyses intimated that alteration of the signaling pathways related to erb-b2 receptor tyrosine kinase 4 (ERBB4) and wnt family member 5A (WNT5A), and bone morphogenetic protein (BMP) may be associated with the TCDD-induced thymus atrophy. We also observed significantly altered expression levels of genes including interleukine 13 receptor subunit alpha 2 (IL13RA2), transforming growth factor beta 1 (TGFß1), collagen type III alpha 1 chain (COL3A1), and collagen type IX alpha 3 chain (COL9A3), implying immunosuppression, fibrosis development, and collagen deposition. Collectively, these findings suggest that TCDD exposure activates the ckAHR1-ckARNT2 signaling pathway and suppresses immune responses through the prompted differentiation to CD4+CD8-CD25+ and CD4+CD8+CD25+ Tregs and altered expressions of immune-related genes in the thymus of chicken embryos.

Liver transplantation in an adult patient with hepatocellular carcinoma following liver cirrhosis as a complication of the Fontan procedure -A case report.

BACKGROUND: Fontan-associated liver disease (FALD) is a hepatic disorder caused by hemodynamic changes and systemic venous congestion following the Fontan procedure. FALD includes liver cirrhosis and hepatocellular carcinoma (HCC), both of which may require liver transplantation (LT). However, the Fontan circulation, characterized by elevated central venous pressure and reduced cardiac output, is a challenging issue for surgeons and anesthesiologists. CASE: We report a living-donor LT for the treatment of HCC. The patient was a 24-year-old male who underwent the Fontan procedure for pulmonary atresia and right ventricle hypoplasia. We focused on maintaining enough blood volume for cardiac output without causing pulmonary edema, as the patient is not well adapted to changes in volume. Owing to a multidisciplinary approach, the surgery was successfully performed without fatal adverse events. CONCLUSIONS: To our knowledge, this is the first case of isolated LT in a recipient who became an adult after having undergone the Fontan procedure.

Fabrication of a Perfusable 3D In Vitro Artery-Mimicking Multichannel System for Artery Disease Models.

Fabrication of a 3D in vitro model that mimics the artery takes an important role in understanding pathological cell behaviors and mechanisms of vascular diseases by proposing an advanced model that can recapitulate a native vessel condition in a controlled manner. Because a model geometry and the structure of cells are significant for the recapitulation of the hemodynamics of arterial and cell functions, it is necessary to mimic geometries and to induce the proper morphology and orientation of the cells when fabricating a model. In this study, smooth muscle cells (SMCs) and endothelial cells (ECs), which were the main elements in the arterial wall, were cocultured in a multichannel device connected with polydimethylsiloxane (PDMS) fluidic chamber modules to parallelly fabricate a pefusable 3D in vitro human artery-mimicking multichannel system. In the coculture model, a circular PDMS channel with a wrinkled-surface guided directionality and contractile morphology to SMCs, and media perfusion induced directionality to a confluent EC layer as in vivo. Protein markers of cells and synthesized extracellular matrices were demonstrated. Because multichannels were connected to a microfluidic module in a device, it was possible to easily control the microenvironmental conditions and to fabricate coculture models in parallel with a single flow system. Coculture models that can be tuned in designs such as diameter, wall shear stress, and geometry of artery disease were constructed by 3D-printed molds to recapitulate various cellular microenvironments and to model vessels effectively. Finally, the effect of wall shear stress on cells was compared using a device with four different degrees of stenosis channels and investigated in parallel.

Lumbar Stability in Healthy Individuals and Low Back Pain Patients Quantified by Wall Plank-and-Roll Test.

BACKGROUND: Low back pain (LBP) has been linked to the degree of lumbar stability, but evaluating lumbar stability has remained a challenge. Previous research has shown that inertial sensors could be used to quantify motor patterns during the wall plank-and-roll (WPR) test, and that LBP may cause deviations in movement from the general motor patterns observed in healthy individuals. OBJECTIVE: To generalize the lumbar motor patterns during the WPR test in healthy individuals, and to analyze the effect of aging and LBP on the motor patterns during the WPR test. DESIGN: A descriptive, exploratory research with a convenience sample. This study is registered at the Clinical Research Information Service (Korea) under public trial registration numbers KCT0002481 and KCT0002533. SETTING: A biomechanics laboratory of a tertiary university hospital. PARTICIPANTS: 57 healthy individuals (23 men 36.7 ± 15.4 years old and 34 women 42.4 ± 17.7 years old) and 17 patients (5 men 48.4 ± 10.9 years old and 12 women 33.7 ± 9.9 years old) with axial LBP. METHODS: Participants performed the WPR test with 2 inertial sensors placed on the thoracic spine and sacrum. Relative angles between the sensors were calculated to quantify and examine lumbar motion in 3 anatomical planes: axial twist, kyphosis-lordosis, and lateral bending. MAIN OUTCOME MEASURES: General motor patterns during the WPR test in healthy participants were examined, stratified based on age, and changes based on age were analyzed. Motor patterns of LBP patients were compared with those from the healthy group. RESULTS: Movement in the kyphosis-lordosis and lateral bending axes showed little variation in healthy participants, whereas in the axial twist axis there were 2 dominant patterns. A χ 2 test revealed that the distributions of 2 motor patterns in the axial twist axis between the younger group and the older group were significantly different (P < .05). Furthermore, the older group had decreased lordosis at the static position (P = .02) and at the maximal rotating position (P = .03). Compared with the healthy group, LBP patients showed increasing lateral bending at the maximal rotating position (P = .007) and increased lateral bending excursion angle (P = .04) during the WPR test. CONCLUSIONS: A general lumbar motor pattern was observed during the WPR test in the healthy participants, but age contributed to variations in this general pattern. Comparison of motor patterns between healthy individuals and LBP patients revealed a different type of variation in the LBP patients. The results presented should be scrutinized with further research, characterizing specific variations in different subgroups of LBP patients. LEVEL OF EVIDENCE: III.

Development of a pressure sensor system for unobtrusive monitoring of abdominal muscle activities.

Surface electromyogram (sEMG) is often used by to objectively measure muscular activity during rehabilitation exercises. sEMG is accurate, but it is unsuitable for uses outside the clinic, and patients can benefit from an unobtrusive device which can be readily used to ubiquitously measure abdominal muscle activation. In this study, we present a pressure sensor system which can be latched onto a belt to measure abdominal muscle activation. sEMG and pressure sensor output were measured in 15 healthy young males during isometric trunk flexion exercise (public trials registration number, KCT0002351), and the results were highly correlated (median R > 0.939). As initial contact force can change the pressure sensor sensitivity, the experiment was performed at two different levels of belt tightness, but the correlations did not significantly improve after tightening the belt, suggesting that the system can be used to ubiquitously and unobtrusively monitor abdominal muscle activity with minimal discomfort.

A fully integrated microdevice for biobarcode assay based biological agent detection.

An integrated microdevice, consisting of a micropump, a passive mixer, a magnetic separation chamber, and a microcapillary electrophoretic channel, was constructed for biobarcode assay based multiplex biological agent detection in a sample-to-answer-out manner within 30 min with high sensitivity.

Lipopolysaccharide of Aggregatibacter actinomycetemcomitans induces the expression of chemokines MCP-1, MIP-1α, and IP-10 via similar but distinct signaling pathways in murine macrophages.

Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium frequently isolated from lesions of patients with localized aggressive periodontitis. Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, stimulates innate immune cells via Toll-like receptor 4 (TLR4) to initiate inflammatory responses. In this study, we purified LPS from A. actinomycetemcomitans (AaLPS) and investigated its ability to induce the expression of chemokines, which play an important role in recruitment of leukocytes to the infection site. AaLPS induced the expression of chemokines, MCP-1, MIP-1α, and IP-10 in murine macrophages, leading to the infiltration of peripheral blood mononuclear cells in a transwell system. Although TLR4 was essential for the induction of all these chemokines by AaLPS, MCP-1 and MIP-1α expressions were MyD88-dependent, but IP-10 expression was MyD88-independent, as determined using macrophages from mice deficient in TLR4 or MyD88. Furthermore, the activation of ERK and JNK were necessary for the expression of MCP-1 and MIP-1α, whereas p38 MAP kinase and JNK activations were required for IP-10 expression. In addition, IFN-ß/STAT1 signaling was exclusively involved in IP-10 expression but not in MCP-1 or MIP-1α expression. AaLPS also activated the transcription factors, NF-κB, AP-1, NF-IL6, and ISRE, all of which are involved in chemokine gene expression. These results suggest that AaLPS induces the expression of chemokines MCP-1, MIP-1α, and IP-10 through TLR4 in murine macrophages. Further, the induction of MCP-1 and MIP-1α requires MyD88, ERK, and JNK, whereas the induction of IP-10 requires JNK, p38 MAP kinase, and IFN-ß/STAT1.

Fish bone migration to the urinary bladder after rectosigmoid colon perforation.

Fish bones are the most common foreign objects leading to bowel perforation. Most cases are confined to the extraluminal space without penetration of an adjacent organ. However, abscess formation due to the perforation of the rectosigmoid colon by a fish bone can lead to the penetration of the urinary bladder and may subsequently cause the fish bone to migrate into the urinary bladder. In the presented case, a 42-year-old female was admitted for lower abdominal pain. The computed tomography (CT) demonstrated a 5 cm pelvic abscess containing a thin and curvilinear foreign body. After conservative management, the patient was discharged. After 1 mo, the subject developed a mechanical ileus. Surgery had to be delayed due to her hyperthyroidism. Migration of the foreign body to the urinary bladder was shown on additional CT. A Yellowish fish bone 3.5 cm in size was removed through intra-operative cystoscopy. The patient was discharged 8 d after the operation without any unexpected event.

Combination of biobarcode assay with on-chip capillary electrophoresis for ultrasensitive and multiplex biological agent detection.

Early diagnosis of biological agents is of paramount importance to prevent the casualties and fatal disease in human during bioterrorism or biological warfare. In this study, we reported an efficient and sensitive multiplex biological agent detection method based on the DNA biobarcode assay and the micro-capillary electrophoresis (µCE) technology. Monoplex as well as multiplex pathogen identification was performed using five targets including Bacillus anthracis, Francisella tularensis, Yersinia pestis, Vaccinia virus and Botulinum toxin A. Through the DNA biobarcode assay process, the magnetic microparticle-pathogen-polystyrene microbead complexes were formed, and the FAM labeled single stranded barcode DNA could be released from the complexes upon denaturation. Different lengths of a barcode DNA were designed to designate each pathogen, so that the specific peak elution time in the capillary electrophoresis on a chip allows us to distinguish the target with high accuracy within 3 min. We improved the assignment accuracy of the peak in the electropherogram by adding two bracket ladders. Owing to the abundant amount of barcode DNAs, the presence of B. anthracis, F. tularensis, Y. pestis, Vaccinia virus was confirmed with a limit of detection of 50CFU/mL, while Botulinum toxin A was analyzed even at a concentration of 12.5 ag/mL. Multiple pathogen detection was also successfully conducted in a phosphate buffered saline (PBS) as well as a serum medium with background of other pathogens. Thus, our analytical platform based on the biobarcode assay and on-chip CE analysis provides rapid, sensitive, multiplex, and accurate biological agent identification.