Dual aptamer assay for detection of Acinetobacter baumannii on an electromagnetically-driven microfluidic platform.

Rapid detection of Acinetobacter baumannii (AB) is critical for limiting healthcare-associated infections and providing the best treatment for infected individuals. Herein an integrated microfluidic device for AB diagnosis utilizing a new dual aptamer assay was developed for point-of-care (POC) applications; magnetic beads coated with AB-specific aptamers were used to capture bacteria, and quantum dots (QD) bound to a second aptamer were utilized to quantify the amount of bacteria with a light-emitting diode (LED)-induced fluorescence module integrated into the device. Within a rapid detection of 30 min, a limit of detection of only 100 colony-forming units (CFU)/reaction was obtained, and all necessary microfluidic devices were actuated by a combination of permanent magnets and electromagnets. The pumping rate of the micropump was 270 µL/min at only 10 V, which is amenable for POC applications with lower power consumption, and only 10 µL of sample and reagents were required. Given these attributes, an automatic POC device was demonstrated which could perform a dual aptamer assay to diagnose AB by using electromagnetically-driven microfluidic system. This system provides a rapid, sensitive, low power and reagents consumption and fully automated for AB detection by using a dual aptamer assay. It will allow rapid clinical diagnosis of AB in the near future.

A structure-free digital microfluidic platform for detection of influenza a virus by using magnetic beads and electromagnetic forces.

H1N1, a subtype of influenza A virus, has emerged as a global threat in the past decades. Due to its highly infectious nature, an accurate and rapid detection assay is urgently required. Therefore, this study presents a new type of digital microfluidic platform for H1N1 virus detection by utilizing a one-aptamer/two-antibodies assay on magnetic beads. The droplets containing magnetic beads were driven by electromagnetic forces on a structure-free, super-hydrophobic surface to automate the entire assay within 40 min. With different levels of hydrophobic modification, the droplets could be easily controlled and positioned without any assisted microstructure. The tunable electromagnetic forces could be adjusted for three kinds of operating modes for the manipulations of beads and droplets, including movement of droplets containing magnetic beads, mixing of two droplets and beads extraction out of droplets. When compared with previous studies, the manipulations of droplets and magnetic particles in this study are more flexible as they can be easily adjusted by fine-tuning the magnetic flux density. Furthermore, the magnetic beads also served as three-dimensional substrates for the new enzyme-linked immunosorbent assay (ELISA)-like assay. The magnetic beads were conjugated with aptamers, which have high specificity towards H1N1 viruses such that they could be specifically captured and detected. The horseradish peroxidase-conjugated secondary antibody was then used to activate tyramide-tetramethylrhodamine (TTMR) such that fluorescent signals could be amplified. With this approach, the limit of detection was experimentally found to be 0.032 hemagglutination units/reaction, which is sensitive enough for clinical diagnostics. This kind of digital microfluidic platform with the ELISA-like assay could effectively reduce the consumption of samples and reagents such that the volume of all droplets including the H1N1 sample, antibodies, TTMR and wash buffers was only 20 µL. This is the first time that a digital microfluidic platform was demonstrated such that the entire diagnostic process for influenza A H1N1 viruses could be performed by using electromagnetic forces, which could be promising for rapid and accurate diagnosis of influenza.

A sample-to-answer, portable platform for rapid detection of pathogens with a smartphone interface.

Emerging and re-emerging infectious diseases pose global threats to human health. Although several conventional diagnostic methods have been widely adopted in the clinic, the long turn-around times of "gold standard" culture-based techniques, as well as the limited sensitivity of lateral-flow strip assays, thwart medical progress. In this study, a smartphone-controlled, automated, and portable system was developed for rapid molecular diagnosis of pathogens (including viruses and bacteria) via the use of a colorimetric loop-mediated isothermal amplification (LAMP) approach on a passive, self-driven microfluidic device. The system was capable of 1) purifying viral or bacterial samples with specific affinity reagents that had been pre-conjugated to magnetic beads, 2) lysing pathogens at low temperatures, 3) executing isothermal nucleic acid amplification, and 4) quantifying the results of colorimetric assays for detection of pathogens with an integrated color sensor. The entire, 40 min analytical process was automatically performed with a novel punching-press mechanism that could be controlled and monitored by a smartphone. As a proof of concept, the influenza A (H1N1) virus and methicillin-resistant Staphylococcus aureus bacteria were used to characterize and optimize the device, and the limits of detection were experimentally found to be 3.2 × 10-3 hemagglutinating units (HAU) per reaction and 30 colony-forming units (CFU) per reaction, respectively; both such values represent high enough sensitivity for clinical adoption. Moreover, the colorimetric assay could be both qualitative and quantitative for detection of pathogens. This is the first instance of an easy-to-use, automated, and portable system for accurate and sensitive molecular diagnosis of either viruses or bacteria, and it is envisioned that this smartphone-controlled apparatus may serve as a platform for clinical, point-of-care pathogen detection, particularly in resource-limited settings.

An integrated microfluidic system for on-chip enrichment and quantification of circulating extracellular vesicles from whole blood.

Circulating extracellular vesicles (EVs), which can contain a wide variety of molecules such as proteins, messenger ribonucleic acids (mRNAs), micro ribonucleic acids (miRNAs) and deoxyribonucleic acids (DNAs) from cells or tissues of origin, have attracted great interest given their potential to serve as biomarkers that can be harvested in body fluids (i.e., relatively non-invasive). Since enrichment and detection of circulating EVs from whole blood have proven challenging, we report herein a fully integrated microfluidic system combining a membrane-based filtration module (i.e. pneumatically-driven microfluidic devices) and a magnetic-bead based immunoassay capable of automating blood treatment, EV enrichment, and EV quantification directly from human whole blood. Three functional modules were implemented; the first, a stirring-enhanced filtration module for separating plasma from blood cells, was characterized by a plasma recovery rate of 65%, a filtrate flow rate of 22 µL min-1, and a vesicle recovery rate of 94% within only 8 min (using 500 µL of blood). The second module, a magnetic bead-based EV enrichment device for immunocapture of circulating EVs from plasma, was characterized by a capture rate of 45%. The final module performed an on-chip enzyme-linked immunosorbent assay for plasma EV quantification in plasma. Given the automated capacity of this system, it could show promise in circulating EV research and clinical point-of-care applications.

The Special Considerations in the Surgical Management of Proximal Anterior Cerebral Artery Aneurysms.

BACKGROUND: Proximal anterior cerebral artery (A1) aneurysms are difficult to clip because of their frequent proximity to perforators, location behind the parent artery, or adherence to surrounding structures. METHODS: We retrospectively reviewed a consecutive series of patients with A1 aneurysms and report the clinical status, radiologic findings, treatment methods, and outcome. RESULTS: This series included 19 male and 12 female patients with a mean age of 50 years. The morphology of the A1 aneurysms was fusiform in 2 patients and saccular in the remaining 29 patients. Multiple aneurysms were presented in 9 patients (29.0%). On admission, 26 patients (83.9%) presented with subarachnoid hemorrhage, 3 of whom had an additional intracerebral hematoma. All surgeries were performed with a standard pteriomal craniotomy. The mean Glasgow Outcome Scale score at final follow-up was 4.8 (interquartile range, 5, 5), with 26 patients (83.9%) rated as 5. The mean follow-up time was 38.5 months (range, 12-60 months). CONCLUSIONS: A1 aneurysms are rare but have their own complex characteristics and are difficult to treat. Meticulous analysis of the relevant angiographs is needed for their diagnosis. An important consideration in surgery is the preservation of perforators and prevention of rupture. Wide opening of the sylvian fissure and temporary control of the parent artery can facilitate dissection of the A1 aneurysms dome. Multiple intraoperative monitoring methods, such as microvascular Doppler ultrasonography and somatosensory and motor evoked potential monitoring, can reduce the relevant complications of surgery.

A microfluidic platform integrated with field-effect transistors for enumeration of circulating tumor cells.

Circulating tumor cells (CTCs) are one of the promising cancer biomarkers whose concentrations are measured not only in the initial diagnostic stages, but also as treatment progresses. However, the existing methods for CTC detection are relatively time-consuming and labor-intensive. In this study, a new microfluidic platform integrated with field-effect transistors (FETs) and chambers for the trapping of CTCs was developed. This novel design could not only trap CTCs from whole blood samples, but also enumerate them via FET sensing of CTC-specific aptamer-CTC complexes. The FET output signal was experimentally found to increase with the increasing number of captured CTCs. More importantly, the enumeration of spiked CTCs in blood samples could be achieved in accordance with the signals measured on the FET devices. We therefore believe that this automated system could be a useful tool for enumeration of CTCs.

A nitrocellulose membrane-based integrated microfluidic system for bacterial detection utilizing magnetic-composite membrane microdevices and bacteria-specific aptamers.

Bacteria such as Acinetobacter baumannii (AB) can cause serious infections, resulting in high mortality if not diagnosed early and treated properly; there is consequently a need for rapid and accurate detection of this bacterial species. Therefore, we developed a new, nitrocellulose-based microfluidic system featuring AB-specific aptamers capable of automating the bacterial detection process via the activity of microfluidic devices composed of magnetic-composite membranes. Electromagnets were used to actuate these microfluidic devices such that the entire diagnostic process could be conducted in the integrated microfluidic system within 40 minutes with a limit of detection as low as 450 CFU per reaction for AB. Aptamers were used to capture AB in complex samples on nitrocellulose membranes, and a simple colorimetric assay was used to estimate bacterial loads. Given the ease of use, portability, and sensitivity of this aptamer-based microfluidic system, it may hold great promise for point-of-care diagnostics.

Digital quantification of DNA via isothermal amplification on a self-driven microfluidic chip featuring hydrophilic film-coated polydimethylsiloxane.

Loop-mediated isothermal amplification (LAMP) is a DNA amplification approach characterized by high sensitivity and specificity. In "digital LAMP", small quantities of both template DNA and reagents are encapsulated within a droplet or microwell, allowing for analysis of precious nucleic acid samples in shorter amounts of time relative to traditional DNA amplification protocols (e.g., PCR) with an improved limit of detection. In this study, an integrated, self-driven microfluidic chip was designed to carry out digital LAMP. The entire quantification process could be automatically performed on this chip via capillary forces enabled through microwells comprised of polydimethylsiloxane (PDMS) surfaces coated with a hydrophilic film; no external pumps were required. Moreover, digitized droplets could be separated from each other by normally-closed microvalves. The contact angle of the hydrophilic film-coated PDMS surface was only 14.3°. This is the first time that a rapid (30min) and simple method has been used to create hydrophilic PDMS surfaces that allow for digital LAMP to be performed in a self-driven microfluidic device. As a proof of concept, amplification of a gene specific to a vancomycin-resistant Enterococcus strain was performed on the developed microfluidic chip within 30min, and the limit of detection was only 11 copies with a volume of 30µL. This device may therefore become a promising tool for clinical diagnosis and point-of-care applications.

A microfluidic chip capable of generating and trapping emulsion droplets for digital loop-mediated isothermal amplification analysis.

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that rapidly amplifies specific DNA molecules at high yield. In this study, a microfluidic droplet array chip was designed to execute the digital LAMP process. The novel device was capable of 1) creating emulsion droplets, 2) sorting them into a 30 × 8 droplet array, and 3) executing LAMP across the 240 trapped and separated droplets (with a volume of 0.22 nL) after only 40 min of reaction at 56 °C. Nucleic acids were accurately quantified across a dynamic range of 50 to 2.5 × 103 DNA copies per µL, and the limit of detection was a single DNA molecule. This is the first time that an arrayed emulsion droplet microfluidic device has been used for digital LAMP analysis. When compared to microwell digital nucleic acid amplification assays, this droplet array-based digital LAMP assay eliminates the constraint on the size of the digitized target, which was determined by the dimension of the microwells for its counterparts. Moreover, the capacity for hydrodynamic droplet trapping allows the chip to operate in a one-droplet-to-one-trap manner. This microfluidic chip may therefore become a promising device for digital LAMP-based diagnostics in the near future.

[Surgical management of proximal anterior cerebral artery (A1) aneurysms].

OBJECTIVE: To review our experience in surgical management of proximal anterior cerebral artery (A1) aneurysms in 23 patients. METHODS: Between January, 2004 and December, 2014, 23 patients (1.6%) with A1 aneurysms diagnosed by CTA or DSA were treated surgically. The "3H" therapy was adopted for postoperative prevention of cerebrovascular spasm. All the patients were followed up and examined with cerebrovascular CTA at 6, 12, 48 and 60 months after the operation with their Glasgow Outcome Scale score recorded. RESULTS: The patients consisted of 15 men and 8 women with an age range of 16 to 72 years (mean 51.3 years). The average diameter of the aneurysms was 5.8 mm, ranging from 3.2 to 9.7 mm. Twenty-two saccular aneurysms were found in these patients; 21 patients presented with SAH and two had vascular malformation. All the A1 aneurysms were managed through the pterional approach, and the mean postoperative Glasgow Outcome Scale score was 4.8. CONCLUSION: Thorough analysis of the angiographic data is essential for the diagnosis and treatment of A1 aneurysms. Preservation of the perforators and prevention of aneurysm rupture are critical during the surgery. Full exposure of the Sylvian fissure and temporary occlusion of the parent artery ensures safe and effective dissection of A1 aneurysms.

Pyrogallol abates VSMC migration via modulation of Caveolin-1, matrix metalloproteinase and intima hyperplasia in carotid ligation mouse.

Migration of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia and other vascular diseases. Caveolin-1 (Cav-1) has been recognized as a proliferative inhibitor of VSMCs and is likely to be an important regulator of VSMC migration. The underlying mechanism of pyrogallol on the VSMC migration is not fully understood. This study attempted to dissect the role of Cav-1 and matrix metalloproteinase (MMP) in VSMC migration and to investigate the effect of pyrogallol on VSMC mobility during carotid artery ligation mice. The mRNA expression of MMP-3 and MMP-13 was down-regulated in cultured VSMC prepared from Cav-1-deficient (Cav-1 KO) mice whereas MMP-14 expression was up-regulated. Pyrogallol effectively inhibited the migration of Cav-1 KO VSMC by promoting the expression of tissue inhibitors of metalloproteinase (TIMP)-2. Pyrogallol also inhibited the migration of Cav-1 wild type (WT) VSMC, however, by increasing TIMP-1 expression and repressing MMP-2 activity. In a parallel in vivo study, intra-peritoneal (ip) of pyrogallol to carotid artery ligated mice significantly suppressed intima formation in mice carotid artery. Furthermore, the proMMP-9 activity in pyrogallol-treated mice serum significantly increased from Day 0 to Day 2 and decreased from Day 2 to Day 7 in a time-dependent manner. In addition, WT mice treated with pyrogallol had significantly reduced neointima formation, whereas no differences were observed in Cav-1 knock out (KO) mice. These results suggest that pyrogallol not only inhibited VSMC migration but also effectively diminishes the severity of neointima hyperplasia, implying that pyrogallol possesses potential anti-atherogenic effects for the treatment of vascular diseases.

Increased HDAC3 and decreased miRNA-130a expression in PBMCs through recruitment HDAC3 in patients with spinal cord injuries.

The study was performed to investigate the molecular mechanism for SCI patients. The interaction between miRNA-130a and HDAC was demonstrated in PBMCs from SCI patients. Increased HDAC3 and decreased miRNA-130a were observed in PBMCs from AS patients. Next, HDAC3 loss-of-function or HAAC3 inhibition promoted the expression of miRNA-130a, and HDAC3 could be recruited to the promoter region of the gene, miRNA-130a, in PBMCs. In addition, linear regression analysis indicated that mRNA expression results were highly negative correlated between HDAC3 and miRNA-130a in PBMCs from SCI patients. Furthermore, miRNA-130a down expression increased the expression of HDAC3 in PBMCs. Loss-of-function of miRNA-130a promoted PBMCs apoptosis, but HDAC3 loss-of-function had no significant effect on the apoptotic cell. In addition, miR-130a overexpression decreased, whereas miR-130a inhibition increased, the expression of TNF-α in PBMCs. Furthermore, HDAC3 loss-of-function or HAAC3 inhibition associated with simultaneous up-regulation the expression of miR-130a and down-regulation the expression of TNF-α in PBMCs. In conclusion, HDAC3 regulated a distinct underlying molecular and pathogenic mechanism of SCI by forming a negative feedback loop with miR-130a and enhanced TNF-1α expression.

[Individualized endovascular treatment of cerebral venous thrombosis: analysis of 168 patients].

OBJECTIVE: To investigate the effects of individualized endovascular treatment on cerebral venous thrombosis of different types. METHODS: 168 patients with cerebral venous thrombosis underwent individualized endovascular treatment: direct thrombolysis via internal jugular vein in 26 cases, injection of urokinase via common carotid artery in 98 cases, stent angioplasty in venous sinus in 9, simple anticoagulant therapy in 20 cases, and treating combined intracranial hemorrhage simultaneously in 15 cases. Follow-up was conducted for 6-168 months. RESULTS: Follow-up showed that the effective rate was 97.6%, recurrence rate was 6.6%, complication incidence rate was 1.2%, and death rate was 0.6%. Venous sinus recanalization occurred and primary drainage rebounded in most eases. Lateral drainage was strengthened in some cases whose venous sinuses did not recover ideally. The symptoms of most cases were alleviated along with the decrease of intracranial pressure. CONCLUSION: Individualized endovascular treatment based on the characteristics of the disease is effective in treatment of cerebral venous thrombosis.