Gravity-driven flow control in a fully integrated microfluidic cartridge for molecular point-of-care testing.

Molecular point-of-care testing (POCT) system is crucial for the timely prevention and control of infectious diseases. We recently proposed a gravity-driven microfluidic cartridge for molecular POCT detection, without the need for external sources or actuators, demonstrating the advantages in terms of the reduced cartridge size and low development costs. How to achieve precise control of liquid flow behavior is challenging for the gravity-driven cartridge. In this work, we explored the underlying mechanism of flow control in the cartridge and offered optimized solutions for our cartridge design to achieve precise control of dynamic flow rates and enhance pumping efficiency significantly. Through the computational fluid dynamics simulations, we demonstrated that adopting an asymptotic contraction chamber geometry design and a closed-loop air flow channel design with the cartridge inlet can facilitate stable laminar flow of the liquid in our microfluidic cartridge, enabling precise control of flow velocity. We further optimized the microchannel diameter and the contact angle of the liquid with the microchannel wall. The effectiveness of the optimized cartridge for POCT detection was well validated by the accurate detection of the human papillomavirus type 16 virus in the 120 clinical swab samples.

Patterned Photonic Nitrocellulose for Pseudopaper ELISA.

We report an enzyme-link immunosorbent assay (ELISA) based on patterned pseudopaper that is made of photonic nitrocellulose for highly sensitive fluorescence bioanalysis. The pseudopaper is fabricated using self-assembled monodisperse SiO2 nanoparticles that are patterned on a polypropylene substrate as template. The self-assembled nanoparticles have a close-packed hexagonal (opal) structure, so the resulting nitrocellulose has a complementary (inverse opal) photonic structure. Owing to the slow-photon effect of the photonic structure, fluorescence emission for ELISA is enhanced by up to 57-fold without increasing the assay time or complexity. As the detection signal is significantly amplified, a simple smartphone camera suffices to serve as the detector for rapid and on-site analysis. As a demonstration, human IgG is quantitatively analyzed with a detection limit of 3.8 fg/mL, which is lower than that of conventional ELISA and paper-based ELISA. The consumption of sample and reagent is also reduced by 33 times compared with conventional ELISA. Therefore, the pseudopaper ELISA based on patterned photonic nitrocellulose is promising for sensitive, high-throughput bioanalysis.

Paper-based colorimetric glucose sensor using Prussian blue nanoparticles as mimic peroxidase.

A novel paper-based colorimetric glucose sensor was proposed employing Prussian blue nanoparticles (PB NPs) as mimic peroxidase. The sensor was manufactured by spraying solution containing PB NPs, glucose oxidase and chromogenic agents into a paper, then coating the filter layer and spreading layer on the top. The layer-by-layer structure enabled the sensor detect glucose in whole blood, as well as elimination of the coffee-ring effect which ensure the performance. As a powerful alternative to natural peroxidase, PB NPs showed the mimic enzymatic activity well preserved in dry environment. The manufacture process of the sensor is easy to be industrialized. Under optimal conditions, the sensor exhibited a linear range from 2.5 mM to 25 mM for glucose in blood with satisfactory reproducibility (the coefficient of variant <4%), great storage stability (1 month at 45 °C) and excellent linearity compared with those commercial kits (R > 0.99). Coupled with a handhold device, the PB NPs-based test strip realized the goal of personal operation, user-friendly control, automatic readouts, and data storage, and able to link the Cloud, showing unique potential in clinical application, especially in community-level medical scenarios.

A sample-to-answer, quantitative real-time PCR system with low-cost, gravity-driven microfluidic cartridge for rapid detection of SARS-CoV-2, influenza A/B, and human papillomavirus 16/18.

The pandemic of coronavirus disease 2019 (COVID-19), due to the novel coronavirus (SARS-CoV-2), has created an unprecedented threat to the global health system, especially in resource-limited areas. This challenge shines a spotlight on the urgent need for a point-of-care (POC) quantitative real-time PCR (qPCR) test for sensitive and rapid diagnosis of viral infections. In a POC system, a closed, single-use, microfluidic cartridge is commonly utilized for integration of nucleic acid preparation, PCR amplification and florescence detection. But, most current cartridge systems often involve complicated nucleic acid extraction via active pumping that relies on cumbersome external hardware, causing increases in system complexity and cost. In this work, we demonstrate a gravity-driven cartridge design for an integrated viral RNA/DNA diagnostic test that does not require auxiliary hardware for fluid pumping due to adopted extraction-free amplification. This microfluidic cartridge only contains two reaction chambers for nucleic acid lysis and amplification respectively, enabling a fast qPCR test in less than 30 min. This gravity-driven pumping strategy can help simplify and minimize the microfluidic cartridge, thus enabling high-throughput (up to 12 test cartridges per test) molecular detection via a small cartridge readout system. Thus, this work addresses the scalability limitation of POC molecular testing and can be run in any settings. We verified the analytical sensitivity and specificity of the cartridge testing for respiratory pathogens and sexually transmitted diseases using SARS-CoV-2, influenza A/B RNA samples, and human papillomavirus 16/18 DNA samples. Our cartridge system exhibited a comparable detection performance to the current gold standard qPCR instrument ABI 7500. Moreover, our system showed very high diagnostic accuracy for viral RNA/DNA detection that was well validated by ROC curve analysis. The sample-to-answer molecular testing system reported in this work has the advantages of simplicity, rapidity, and low cost, making it highly promising for prevention and control of infectious diseases in poor-resource areas.

Multiple chemiluminescence immunoassay detection of the concentration ratio of glycosylated hemoglobin A1c to total hemoglobin in whole blood samples.

The concentration ratio of glycosylated hemoglobin A1c (HbA1c) to total hemoglobin (Hb) has long been used to accurately determine stagewise diabetes because this parameter represents a reliable and accurate biomarker of mean 90-day blood glucose values. In this paper, we report a time-resolved chemiluminescence assay that can detect both Hb and HbA1c. For the determination of Hb, the interaction of heme in Hb with H2O2 in NaOH solution was performed to generate a chemiluminescence peak. HbA1c was detected using a sandwich immunoassay based on an acridine ester-labeling method using the same Hb chemiluminescence trigger system. The results showed that the repeatability %CV of the proposed method for multiple detections of HbA1c and Hb ranged from 1.22 to 2.21%, with a median value of 1.73%, while the within-site reproducibility %CV ranged from 2.13 to 3.27%, with a median value of 2.81%. Compared with the conventional HPLC method (BIO-RAD D10 system), the correlation coefficient was 0.9959. In conclusion, a time-resolved multiple chemiluminescence immunoassay biosensor for HbA1c/Hb detection was established, and the method has excellent reproducibility and accuracy, thus demonstrating great potential for clinical application.

A New Hematocrit Measurement Method Using a Chemiluminescence Biosensor and Its Application in a Chemiluminescence Immunoassay Platform for Myocardial Markers Detection with Whole Blood Samples.

The accuracy and precision of analyte concentrations measured in whole blood by chemiluminescence immunoassay (CLIA) have been significantly affected by erythrocytes, which leads to poor application of whole blood CLIA in clinical practice. In this work, a chemiluminescence biosensing optical platform for blood hematocrit (HCT) analysis using MAGICL 6000 (Getein Biotechnology, Nanjing, China) was designed, implemented, and fully characterized. The developed method was successfully applied to determine various HCT levels of human blood from 0% to 65%, with a correlation coefficient of 0.9885 compared with the conventional method (Sysmex XE 5000, Kobe, Japan). A mathematical model was developed to quantitatively evaluate the impact of HCT on the results of two sample types (whole blood vs. plasma). Combining the established HCT method and mathematical model with CLIA on MAGICL 6000, the precision was significantly improved by almost 20%. Comparison studies using whole blood samples and corresponding plasma samples showed that the square of the correlation coefficients of troponin I (cTnI), myoglobin (MYO), creatine kinase MB (CK-MB), and N-terminal pro-hormone brain natriuretic peptide (NT-proBNP) were increased to 0.9992, 0.9997, 0.9996, and 0.9994, respectively, showing a great potential for clinical application.

Ultrasensitive chemiluminescence immunoassay with enhanced precision for the detection of cTnI amplified by acridinium ester-loaded microspheres and internally calibrated by magnetic fluorescent nanoparticles.

A novel enhanced chemiluminescent immunoassay (CLIA) for ultrasensitive and excellent precisive determination of cardiac troponin I (cTnI) was reported. The method made full use of poly[(N-isopropyl acrylamide)-co-(methacrylic acid)] (P(NIPAM-co-MAA)) microspheres as new potential signal enhancers and magnetic fluorescent nanoparticles as internal standards for better precision. This protocol involved a sandwich format, in which the antigen in the sample was captured by the immobilized antibodies on the surface of magnetic fluorescent beads and recognized by the other antibodies labeled with acridinium ester (AE)-loaded P(NIPAM-co-MAA) microspheres. The combination of the remarkable sensitivity of the enhanced CLIA method and the use of P(NIPAM-co-MAA) microspheres as anti-cTnI carriers for acridinium ester signal amplification provided an extremely sensitive limit of blank (LoB) at 0.097 pg mL-1, a limit of detection (LoD) at 0.116 pg mL-1, and a limit of quantitation (LoQ) at 0.606 pg mL-1, much greater than those achieved by the classical chemiluminescence immunoassay (CLIA, Getein). Moreover, the intra-day variable coefficient can be improved to 1.21-2.12%, and inter-day variability was 2.01-3.49% under the application of magnetic fluorescent beads as an internal standard. The sensitivity and precision have reached a high level, comparable with the current commercial detection kits. The results showed a good correlation with a commercial chemiluminescence assay (CLIA, Abbott), with a correlation coefficient of 0.9883. This proposed method has been successfully applied to the clinical determination of cTnI in the human serum.

Eight biomarkers on a novel strip for early diagnosis of acute myocardial infarction.

Accurate detection of markers in human serum is important in the early diagnosis of acute myocardial infarction (AMI). This work presents a novel eight biomarker strip, which combines dry chemistry with a fluorescence lateral flow assay. Eight AMI markers were employed simultaneously for sensitive detection, including cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C), uric acid (UA), myoglobin (Myo), creatine kinase-MB (CK-MB), and cardiac troponin I (cTnI). The strip offers the advantages of simple fabrication, convenience, time-saving detection and accurate assessment for AMI. Moreover, the strip possesses acceptable applicability for human serum. This proposed strategy establishes a remarkable platform for the construction of a multi-target detection strip that is feasible for accurate detection for real human serum samples.

Endothelial progenitor cell mediates transport of hepatitis B.

BACKGROUND: Hepatitis B virus (HBV) replication has been reported to be involved in many extrahepatic viral disorders; however, the mechanism by which HBV is transinfected into extrahepatic tissues such as myocardium and causes HBV associated myocarditis remains largely unknown. METHODS: In this study, endothelial progenitor cells (EPCs) were infected by HBV and then transfused into ischemic model of mice. HBV surface and core antigen as well as mutation of HBV particles were detected by immunohistochemistry, fluorescent activated cell sorter and transmission electron microscopy in vitro and in vivo. RESULTS: Human cord blood EPCs, but not human umbilical vein endothelial cells (HUVECs) could be effectively infected by taking up HBV in vitro. HBV envelope surface and core antigen expressions were first detectable in EPCs at day 3 after virus challenge, sustained for up to 11 days, and decreased thereafter. Similarly, the virus particles were the most abundant in EPCs in the first week observed by a transmission electron microscope, and declined in 3 weeks after HBV infection. HBV DNA but not HBV cccDNA in EPCs were detectable even 3 weeks after virus challenge, as shown by PCR analysis. Furthermore, intravenous transplantation of HBV-treated EPCs into myocardial infarction Sprague & Dawley rats model resulted in incorporation of both EPCs and HBV into injured endothelial tissues of capillaries in the ischemic border zone. CONCLUSIONS: These results strongly support that EPCs serve as virus carrier mediating HBV trans-infection into the injured myocardial tissues. The findings might suggest a novel mechanism for HBV-associated myocarditis.

Disposable Morpho menelaus Based Flexible Microfluidic and Electronic Sensor for the Diagnosis of Neurodegenerative Disease.

Rapid early disease prevention or precise diagnosis is almost impossible in low-resource settings. Natural ordered structures in nature have great potential for the development of ultrasensitive biosensors. Here, motivated by the unique structures and extraordinary functionalities of ordered structures in nature, a biosensor based on butterfly wings is presented. In this study, a flexible Morpho menelaus (M. menelaus) based wearable sensor is integrated with a microfluidic system and electronic networks to facilitate the diagnosis of neurodegenerative disease (ND). In the microfluidic section, the structural characteristics of the M. menelaus wings up layer are combined with SiO2 nanoparticles to form a heterostructure. The fluorescent enhancement property of the heterostructure is used to increase the fluorescent intensity for multiplex detection of two proteins: IgG and AD7c-NTP. For the electronic section, conductive ink is blade-coated on the under layer of wings for measuring resistance change rate to obtain the frequency of static tremors of ND patients. The disposable M. menelaus based flexible microfluidic and electronic sensor enables biochemical-physiological hybrid monitoring of ND. The sensor is also amenable to a variety of applications, such as comprehensive personal healthcare and human-machine interaction.

Quantitative and ultrasensitive detection of multiplex cardiac biomarkers in lateral flow assay with core-shell SERS nanotags.

Rapid and sensitive quantification of multiplex proteins in a wide concentration range is challenging in high throughput analysis. Herein, we proposed a lateral flow assay (LFA) based on core-shell surface enhanced Raman scattering (SERS) nanotags for multiplex and quantitative detection of cardiac biomarkers for the early diagnosis of acute myocardial infarction (AMI). In practice, Raman dyes (RDs) were embedded into the interior-gap of silver core and gold shell nanoparticles (NPs) to form SERS nanotags as labels instead of gold colloids and three test lines were employed in the strip for the detection of three cardiac biomarkers, Myo, cTnI, and CK-MB, respectively. Due to the amplified signal of the SERS nanotags and the high surface area to volume ratio (SVR) of porous nitrocellulose (NC) membrane, ultrasensitive quantification of protein markers with wide linear dynamic range (LDR) was realized, which is crucial for the quick detection of multiplex biomarkers in the same sample without pretreatments at bedsides. This method makes it possible for LFA in point of care testing (POCT) to be comparable with chemiluminescence immunoassay (CLIA) used in labs.

Infection of hepatitis B virus in extrahepatic endothelial tissues mediated by endothelial progenitor cells.

BACKGROUND: Hepatitis B virus (HBV) replication has been reported to be involved in many extrahepatic viral disorders; however, the mechanism by which HBV is trans-infected into extrahepatic tissues such as HBV associated myocarditis remains largely unknown. RESULTS: In this study, we showed that human cord blood endothelial progenitor cells (EPCs), but not human umbilical vein endothelial cells (HUVECs) could be effectively infected by uptake of HBV in vitro. Exposure of EPCs with HBV resulted in HBV DNA and viral particles were detected in EPCs at day 3 after HBV challenge, which were peaked around day 7 and declined in 3 weeks. Consistently, HBV envelope surface and core antigens were first detected in EPCs at day 3 after virus challenge and were retained to be detectable for 3 weeks. In contrast, HBV covalently closed circular DNA was not detected in EPCs at any time after virus challenge. Intravenous transplantation of HBV-treated EPCs into myocardial infarction and acute renal ischemia mouse model resulted in incorporation of HBV into injured heart, lung, and renal capillary endothelial tissues. CONCLUSION: These results strongly support that EPCs serve as virus carrier mediating HBV trans-infection into the injured endothelial tissues. The findings might provide a novel mechanism for HBV-associated myocarditis and other HBV-related extrahepatic diseases as well.

[Expression of KISS-1 and GnRH in rat hypothalamus].

OBJECTIVE: To assay the expression of KiSS-1 and GnRH in the male rat hypothalamus at different developmental stages, and to explore the significance of KiSS-1 in sex development onset and normal reproduction regulation. METHODS: Expression analyses of KiSS-1 and GnRH genes were conducted in the rat hypothalamus at different developmental stages with RT-PCR and real time-PCR. The testosterone level was assayed by chemoluminescence technique. RESULTS: KiSS-1 mRNA rose gradually during sex development in the rat hypothalamus, highest at puberty and lowered a little at adulthood. KiSS-1 mRNA of the prepubertal, early pubertal, pubertal and adult rats was 1.7, 2.1, 3.5 and 2.0 times higher than that of the infantile rats respectively. The expression of GnRH and KiSS-1 correlated positively (r = 0.905, P < 0.05). But the activation of GnRH neuron was later than KiSS-1. The expression of GnRH was the highest in the puberty rats. GnRH mRNA of the prepubertal, early pubertal, pubertal and adult rats was 1.1, 1.94, 2.42 and 1.92 times higher than that of the infantile rats respectively. The level of testosterone in the adult rats was significantly higher than that at the earlier stage and was the highest at the adult stage. CONCLUSION: The expression of KiSS-1 correlates positively with that of GnRH. KiSS-1 may participate in the regulation of GnRH and is relevant to puberty onset and the regulation of reproduction function.

Transpiration-Inspired Fabrication of Opal Capillary with Multiple Heterostructures for Multiplex Aptamer-Based Fluorescent Assays.

In this work we report a method for the fabrication of opal capillary with multiple heterostructures for aptamer-based assays. The method is inspired by plant transpiration. During the fabrication, monodisperse SiO2 nanoparticles (NPs) self-assemble in a glass capillary, with the solvent gradually evaporating from the top end of the capillary. By a simple change of the colloid solution that wicks through the capillary, multiple heterostructures can be easily prepared inside the capillary. On the surface of the SiO2 NPs, polydopamine is coated for immobilization of aminomethyl-modified aptamers. The aptamers are used for fluorescent detection of adenosine triphosphate (ATP) and thrombin. Owing to fluorescence enhancement effect of the photonic heterstructures, the fluorescent signal for detection is amplified up to 40-fold. The limit of detection is 32 µM for ATP and 8.1 nM for thrombin. Therefore, we believe this method is promising for the fabrication of analytical capillary devices for point-of-care testing.

The complete mitochondrial genome of the Neochauliodes fraternus (Megaloptera: Corydalidae).

The mitochondrial genome of Neochauliodes fraternus (Megaloptera: Corydalidae) is a circular molecule of 15,768 bp in length, containing 37 typical mitochondrial genes: 13 protein-coding genes (PCGs), 2 ribosomal RNAs, 22 transfer RNAs and a non-coding AT-rich region. Its gene order and arrangement are identical to the common type found in most insect mitogenomes. All PCGs start with a typical ATN codon except for the ND1 which uses TTG as its start codon; all PCGs terminate in the common stop codon TAA or TAG, except for the COI, COIII, ND3, ND5, ND4 and CYTB which use single T as their stop codons. The non-coding AT-rich region is 1031 bp long, located between rrnS and tRNA(lle) genes. It contains some structures of repeated motifs and microsatellite-like elements characteristic of the megaloptera.

The Complete mitochondrial genome of the Neochauliodes rotundatus (Megaloptera: Corydalidae).

The mitochondrial genome of Neochauliodes rotundatus (Megaloptera: Corydalidae) is a circular molecule of 15,774 bp in length, containing 37 typical mitochondrial genes: 13 protein-coding genes (PCGs), 2 ribosomal RNAs, 22 transfer RNAs and a non-coding AT-rich region. Its gene order and arrangement are identical to the common type found in most insect mitogenomes. All PCGs start with a typical ATN codon except for the ND1 which uses TTG as its start codon; all PCGs terminate in the common stop codon TAA or TAG, except for the COI, COIII, ND3, ND5, ND4 and CYTB which use single T as their stop codons. The 1061 bp non-coding AT-rich region is located between rrnS and tRNA(lle) genes, containing some structures of repeated motifs and microsatellite-like elements characteristic of the Megaloptera insects.

Selection of HBsAg-Specific DNA Aptamers Based on Carboxylated Magnetic Nanoparticles and Their Application in the Rapid and Simple Detection of Hepatitis B Virus Infection.

Aptamers are short single-stranded DNA or RNA oligonucleotides and can be selected from synthetic combinatorial libraries in vitro. They have a high binding affinity and specificity for their targets. Agarose gels, nitrocellulose membranes, and adsorptive microplates are often used as carriers to immobilize targets in the SELEX (systematic evolution of ligands by exponential enrichment) process, but the subsequent separation step is tedious and time-consuming. Therefore, we used magnetic nanoparticles (MNPs) as carriers to immobilize the target, hepatitis B surface antigen (HBsAg), which is convenient for fast magnetic separation. In this study, we first selected DNA aptamers against HBsAg by immobilizing HBsAg on the surface of carboxylated MNPs. The ssDNA library of each selection round was prepared by asymmetric PCR amplification for the next selection round. To obtain aptamer sequences, the final selected products were purified by gel electrophoresis, then cloned, and sequenced. DNA aptamers that specifically bind to HBsAg were successfully obtained after 13 selection rounds. The selected aptamers were used to construct a chemiluminescence aptasensor based on magnetic separation and immunoassay to detect HBsAg from pure protein or actual serum samples. There was a linear relationship between HBsAg concentration and chemiluminescent intensity in the range of 1-200 ng/mL. The aptasensor worked well even in the presence of interfering substances and was highly specific in the detection of HBsAg in serum samples, with a detection limit 0.1 ng/mL lower than the 0.5 ng/mL limit of an ELISA in use at the hospital. This aptasensor can contribute to better detection of hepatitis B virus infection.

Genetic Variation of BCL2 (rs2279115), NEIL2 (rs804270), LTA (rs909253), PSCA (rs2294008) and PLCE1 (rs3765524, rs10509670) Genes and Their Correlation to Gastric Cancer Risk Based on Universal Tagged Arrays and Fe3O4 Magnetic Nanoparticles.

With the help of Fe3O4 nagnetic nanoparticles as a solid carrier and an excellent tool for separation, six SNP loci (rs2279115 of BCL2 gene, rs804270 of NEIL2 gene, rs909253 of LTA gene, rs2294008 of PSCA gene, rs3765524 and rs10509670 of PLCE1 gene) were selected to evaluate their relation to gastric cancer risk. Using two kinds of functionalized magnetic nanoparticles and universal tagged arrays, the whole operation procedure including genome DNA extraction and SNP genotyping was performed. All genotypes and allele frequencies were calculated in the cases and controls respectively to analyze their association with gastric cancer risk. Totally 200 pathological samples and 134 normal control subjects were collected. The results demonstrated that four SNP loci (rs2279115, rs804270, rs909253 and rs3765524) showed a potential association with gastric cancer risk, and the other two (rs2294008, rs10509670) possessed no difference/association among cases and controls.

Effects of resveratrol on number and activity of endothelial progenitor cells from human peripheral blood.

1. It has been well established that oestrogens can increase the number of endothelial progenitor cells (EPC) by anti-apoptotic effects. Resveratrol, a polyphenolic phytoalexin extracted from grapes and wine, has been reported to act as an oestrogen receptor agonist. We hypothesize that putative phyto-oestrogen may promote EPC proliferation and survival in vitro. 2. Endothelial progenitor cells were isolated from human peripheral blood and identified immunocytochemically. Endothelial progenitor cells were incubated with resveratrol (1, 10, 25 and 50 mmol/L) or control for specified times. Cell proliferation, migration and in vitro vasculogenesis were assayed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay, modified Boyden chamber assay and in vitro vasculogenesis detection, respectively. 3. Resveratrol increased the number of EPC and promoted EPC proliferation, adhesion and migration in a dose- and time-dependent manner. Cell number peaked at 50 mmol/L resveratrol after incubation for 24 h compared with vehicle control (61.3 +/- 5.8 vs 112.8 +/- 7.2, respectively; P < 0.01). 4. Furthermore, cell cycle analysis showed that 50 mmol/L resveratrol significantly increased the S phase and decreased the G(0)/G(1) phase of EPC. In addition, resveratrol increased vascular endothelial growth factor production and further induced vasculogenesis in vitro. 5. In conclusion, resveratrol significantly induces EPC proliferation, migration and further promotes angiogenesis in vitro.