BCSG1 siRNA delivered by lentiviral vector suppressed proliferation and migration of MDA-MB-231 cells.

Breast cancer-specific gene 1 (BCSG1), also referred to as γ-synuclein (SNCG), is highly expressed in human infiltrating breast carcinomas, but not in normal or benign breast tissue. The present study aimed to evaluate the effects of BCSG1 siRNA delivered by lentiviral vector on breast cancer cells and investigate the underlying mechanisms. BCSG1 RNAi lentiviral vector was constructed and transfected into MDA-MB-231 cells. BCSG1 mRNA levels were determined by quantitative polymerase chain reaction analysis. Cell proliferation, migration and apoptosis were evaluated by using the cell counting kit­8, Transwell assay and flow cytometry, respectively, followed by western blotting to determine the relative levels of AKT, extracellular signal­regulated kinase (ERK), p-AKT and p-ERK expression. BCSG1 mRNA levels were significantly reduced in MDA-MB­231 cells following transfection of BCSG1 siRNA delivered by lentiviral vector. Cell migration and proliferation were significantly decreased and the cell cycle was arrested. Western blot analysis indicated that the protein levels of p-AKT and p-ERK were significantly lower in the BCSG1 siRNA-treated groups compared with the control and negative control groups. Therefore, BCSG1 siRNA delivered by lentiviral vector was able to significantly reduce BCSG1 expression, suppress cell migration and proliferation, possibly through the reduction of the protein levels of p-AKT and p-ERK.

Rotation-Driven Microfluidic Disc for White Blood Cell Enumeration Using Magnetic Bead Aggregation.

We recently defined a magnetic bead-based assay that exploited an agglutination-like response for DNA and applied it to DNA-containing cell enumeration using inexpensive benchtop hardware [ J. Am. Chem. Soc. 2012 , 134 ( 12 ), 5689 - 96 ]. Although cost-efficient, the open-well format assay required numerous manual steps, and the magnetic field actuation scheme was not readily adaptable for integration. Here, we demonstrate a low-cost (<$2 in-lab), higher-throughput "pinwheel assay" platform that relies on a combination of a disposable rotation-driven microdisc (RDM), and a simple bidirectional rotating magnetic field (bi-RMF). The assay was transformed into an integrated microfluidic system using a multilayered polyester microfluidic disc created through laser print, cut and laminate fabrication, with fluid flow controlled by rotation speed without any mechanical valves. The RDM accepts four samples that undergo on-chip dilution to five different concentrations that cover the effective concentration range needed for downstream cell counting by pinwheel assay. We show that a bi-RMF is effective for the simultaneous actuation of pinwheel assays in 20 detection chambers. The optimization of the bi-RMF frequencies allows the RDM-based pinwheel assay detect human genomic DNA down to a mass of human genomic DNA (5.5 picograms) that is roughly equal to the mass in a single cell. For proof of principle, enumeration of the white blood cells in human blood samples on the RDM provided data correlating well (C.V. of 10%) with those obtained in a clinical lab. Fusing the cost-effective RDM with a simple bi-RMF provides a promising strategy for automation and multiplexing of magnetic particle-based agglutination assays.

A novel, integrated forensic microdevice on a rotation-driven platform: Buccal swab to STR product in less than 2 h.

This work describes the development of a novel microdevice for forensic DNA processing of reference swabs. This microdevice incorporates an enzyme-based assay for DNA preparation, which allows for faster processing times and reduced sample handling. Infrared-mediated PCR (IR-PCR) is used for STR amplification using a custom reaction mixture, allowing for amplification of STR loci in 45 min while circumventing the limitations of traditional block thermocyclers. Uniquely positioned valves coupled with a simple rotational platform are used to exert fluidic control, eliminating the need for bulky external equipment. All microdevices were fabricated using laser ablation and thermal bonding of PMMA layers. Using this microdevice, the enzyme-mediated DNA liberation module produced DNA yields similar to or higher than those produced using the traditional (tube-based) protocol. Initial microdevice IR-PCR experiments to test the amplification module and reaction (using Phusion Flash/SpeedSTAR) generated near-full profiles that suffered from interlocus peak imbalance and poor adenylation (significant -A). However, subsequent attempts using KAPA 2G and Pfu Ultra polymerases generated full STR profiles with improved interlocus balance and the expected adenylated product. A fully integrated run designed to test microfluidic control successfully generated CE-ready STR amplicons in less than 2 h (<1 h of hands-on time). Using this approach, high-quality STR profiles were developed that were consistent with those produced using conventional DNA purification and STR amplification methods. This method is a smaller, more elegant solution than current microdevice methods and offers a cheaper, hands-free, closed-system alternative to traditional forensic methods.

Multilevel fluidic flow control in a rotationally-driven polyester film microdevice created using laser print, cut and laminate.

This paper presents a simple and cost-effective polyester toner microchip fabricated with laser print and cut lithography (PCL) to use with a battery-powered centrifugal platform for fluid handling. The combination of the PCL microfluidic disc and centrifugal platform: (1) allows parallel aliquoting of two different reagents of four different volumes ranging from nL to µL with an accuracy comparable to a piston-driven air pipette; (2) incorporates a reciprocating mixing unit driven by a surface-tension pump for further dilution of reagents, and (3) is amenable to larger scale integration of assay multiplexing (including all valves and mixers) without substantially increasing fabrication cost and time. For a proof of principle, a 10 min colorimetric assay for the quantitation of the protein level in the human blood plasma samples is demonstrated on chip with a limit of detection of ∼5 mg mL(-1) and coefficient of variance of ∼7%.

The ARTµS: a novel microfluidic CD4+ T-cell enumeration system for monitoring antiretroviral therapy in HIV patients.

We report on a novel and cost-effective microfluidic platform that integrates immunomagnetic separation and cell enumeration via DNA-induced bead aggregation. Using a two-stage immunocapture microdevice, 10 µL of whole blood was processed to isolate CD4+ T-cells. The first stage involved the immuno-subtraction of monocytes by anti-CD14 magnetic beads, followed by CD4+ T-cell capture with anti-CD4 magnetic beads. The super hydrophilic surface generated during polydimethylsiloxane (PDMS) plasma treatment allowed for accurate metering of the CD4+ T-cell lysate, which then interacted with silica-coated magnetic beads under chaotropic conditions to form aggregates. Images of the resulting aggregates were captured and processed to reveal the mass of DNA, which was used to back-calculate the CD4+ T-cell number. Studies with clinical samples revealed that the analysis of blood within 24 hours of phlebotomy yielded the best results. Under these conditions, an accurate cell count was achieved (R(2) = 0.98) when compared to cell enumeration via flow cytometry, and over a functional dynamic range from 106-2337 cells per µL.

A disposable laser print-cut-laminate polyester microchip for multiplexed PCR via infra-red-mediated thermal control.

Infrared (IR)-mediated thermal cycling system, a method proven to be a effective for sub-µL scale polymerase chain reaction (PCR) on microchips, has been integrated with DNA extraction and separation on a glass microchip in a fully integrated micro Total Analysis System by Easley et al., in 2006. IR-PCR has been demonstrated on both glass and PMMA microdevices where the fabrication (bonding) is not trivial. Polyester-toner (PeT) microfluidic devices have significant potential as cost-effective, disposable microdevices as a result of the ease of fabrication (∼$0.25 USD and <10 min per device) and availability of commercial substrates. For the first time, we demonstrate here the thermal cycling in PeT microchips on the IR-PCR system. Undesirable IR absorption by the black-toner bonding layer was eliminated with a spatial filter in the form of an aluminum foil mask. The solution heating rate for a black PeT microchip using a tungsten lamp was 10.1 ± 0.7 °C s(-1) with a cooling rate of roughly -12 ± 0.9 °C s(-1) assisted by forced air cooling. Dynamic surface passivation strategies allowed the successful amplification of a 520 bp fragment of the λ-phage genome (in 11 min) and a 1500 bp region of Azospirillum brasilense. Using a centrosymmetric chamber configuration in a multichamber PeT microchip, homogenous temperature distribution over all chambers was achieved with inter-chamber temperature differences at annealing, extension and denaturing steps of less than ±2 °C. The effectiveness of the multichamber system was demonstrated with the simultaneous amplification of a 390 bp amplicon of human ß-globin gene in five PeT PCR microchambers. The relative PCR amplification efficiency with a human ß-globin DNA fragment ranged from 70% to 90%, in comparison to conventional thermal cyclers, with an inter-chamber standard deviation of ∼10%. Development of PeT microchips for IR-PCR has the potential to provide rapid, low-volume amplification while also integrating PCR with extraction upstream and separation/detection downstream.

Inexpensive, rapid prototyping of microfluidic devices using overhead transparencies and a laser print, cut and laminate fabrication method.

We describe a technique for fabricating microfluidic devices with complex multilayer architectures using a laser printer, a CO2 laser cutter, an office laminator and common overhead transparencies as a printable substrate via a laser print, cut and laminate (PCL) methodology. The printer toner serves three functions: (i) it defines the microfluidic architecture, which is printed on the overhead transparencies; (ii) it acts as the adhesive agent for the bonding of multiple transparency layers; and (iii) it provides, in its unmodified state, printable, hydrophobic 'valves' for fluidic flow control. By using common graphics software, e.g., CorelDRAW or AutoCAD, the protocol produces microfluidic devices with a design-to-device time of ∼40 min. Devices of any shape can be generated for an array of multistep assays, with colorimetric detection of molecular species ranging from small molecules to proteins. Channels with varying depths can be formed using multiple transparency layers in which a CO2 laser is used to remove the polyester from the channel sections of the internal layers. The simplicity of the protocol, availability of the equipment and substrate and cost-effective nature of the process make microfluidic devices available to those who might benefit most from expedited, microscale chemistry.

Rapid patterning of 'tunable' hydrophobic valves on disposable microchips by laser printer lithography.

We recently defined a method for fabricating multilayer microdevices using poly(ethylene terephthalate) transparency film and printer toner, and showed these could be successfully applied to DNA extraction and amplification (Duarte et al., Anal. Chem. 2011, 83, 5182-5189). Here, we advance the functionality of these microdevices with flow control enabled by hydrophobic valves patterned using laser printer lithography. Laser printer patterning of toner within the microchannel induces a dramatic change in surface hydrophobicity (change in contact angle of DI water from 51° to 111°) with good reproducibility. Moreover, the hydrophobicity of the surface can be controlled by altering the density of the patterned toner via varying the gray-scale setting on the laser printer, which consequently tunes the valve's burst pressure. Toner density provided a larger burst pressure bandwidth (158 ± 18 Pa to 573 ± 16 Pa) than could be achieved by varying channel geometry (492 ± 18 Pa to 573 ± 16 Pa). Finally, we used a series of tuned toner valves (with varied gray-scale) for passive valve-based fluidic transfer in a predictable manner through the architecture of a rotating PeT microdevice. While an elementary demonstration, this presents the possibility for simplistic and cost-effective microdevices with valved fluid flow control to be fabricated using nothing more than a laser printer, a laser cutter and a laminator.

Studies on interaction between Vitamin B12 and human serum albumin.

The binding reaction between Vitamin B12 (B12, cyanocobalamin) and human serum albumin (HSA) was investigated by fluorescence quenching, UV-vis absorption and circular dichroism (CD) spectroscopy. Under simulative physiological conditions, fluorescence quenching data revealed that the quenching constants (Ksv) are 3.99 x 10(4), 4.33 x 10(4), 4.76 x 10(4) and 5.16 x 10(4) M(-1) at 292, 298, 304 and 310 K, respectively. The number of binding sites, n is almost constant around 1.0. On the basis of the results of fluorescence quenching the mechanism of the interaction of B12 with HSA has been found to be a dynamic quenching procedure. Thermodynamic parameters DeltaHTheta= -13.38 kJ mol(-1), DeltaSTheta=66.73 J mol(-1) K(-1) were calculated based on the binding constant. These suggested that the binding reaction was enthalpy and entropy driven, and the electrostatic interaction played major role in stabilizing the reversible complex. The binding distance r=5.5 nm between HSA and B12 was obtained according to Förster theory of energy transfer. The effect of B12 on the conformation of HSA was analyzed by synchronous fluorescence and CD spectroscopy. Synchronous spectra indicated that the polarity around the tryptophan (Trp214) residues of HSA was decreased and its hydrophobicity was increased; however, the alpha-helix content of the protein was predominant in the secondary structure but the CD spectra indicated that B12 induced minor conformational changes of HSA.