A 3D paper microfluidic device for enzyme-linked assays: Application to DNA analysis.

A paper microfluidic device capable of conducting enzyme-linked assays is presented: a microfluidic enzyme-linked paper analytical device (µEL-PAD). The system exploits a wash-free sandwich coupling to form beads/analyte/enzyme complexes, which are subsequently added to the vertical flow device composed of wax-printed paper, waxed nitrocellulose membrane and absorbent/barrier layers. The nitrocellulose retains the bead complexes without disrupting the flow, enabling for an efficient washing step. The entrapped complexes then interact with the chromogenic substrate stored on the detection paper, generating a color change on it, quantified with an open-source smartphone software. This is a universal paper-based technology suitable for high-sensitivity quantification of many analytes, such as proteins or nucleic acids, with different enzyme-linked formats. Here, the potential of the µEL-PAD is demonstrated to detect DNA from Staphylococcus epidermidis. After generation of isothermally amplified genomic DNA from bacteria, Biotin/FITC-labeled products were analyzed with the µEL-PAD, exploiting streptavidin-coated beads and antiFITC-horseradish peroxidase. The µEL-PAD achieved a limit of detection (LOD) and quantification <10 genome copies/µL, these being at least 70- and 1000-fold lower, respectively, than a traditional lateral flow assay (LFA) exploiting immobilized streptavidin and antiFITC-gold nanoparticles. It is envisaged that the device will be a good option for low-cost, simple, quantitative, and sensitive paper-based point-of-care testing.

Portable electroanalytical nucleic acid amplification tests using printed circuit boards and open-source electronics.

The realization of electrochemical nucleic acid amplification tests (NAATs) at the point of care (POC) is highly desirable, but it remains a challenge given their high cost and lack of true portability/miniaturization. Here we show that mass-produced, industrial standardized, printed circuit boards (PCBs) can be repurposed to act as near-zero cost electrodes for self-assembled monolayer-based DNA biosensing, and further integration with a custom-designed and low-cost portable potentiostat. To show the analytical capability of this system, we developed a NAAT using isothermal recombinase polymerase amplification, bypassing the need of thermal cyclers, followed by an electrochemical readout relying on a sandwich hybridization assay. We used our sensor and device for analytical detection of the toxic microalgae Ostreopsis cf. ovata as a proof of concept. This work shows the potential of PCBs and open-source electronics to be used as powerful POC DNA biosensors at a low-cost.

Electroanalytical Paper-Based Nucleic Acid Amplification Biosensors with Integrated Thread Electrodes.

Nucleic acid amplification tests (NAATs) are very sensitive and specific methods, but they mainly rely on centralized laboratories and therefore are not suitable for point-of-care testing. Here, we present a 3D microfluidic paper-based electrochemical NAAT. These devices use off-the-shelf gold plasma-coated threads to integrate electroanalytical readouts using ex situ self-assembled monolayer formation on the threads prior to assembling into the paper device. They further include a sandwich hybridization assay with sample incubation, rinsing, and detection steps all integrated using movable stacks of filter papers to allow time-sequenced reactions. The devices use glass fiber substrates for storing recombinase polymerase amplification reagents and conducting the isothermal amplification. We used the paper-based device for the detection of the toxic microalgae Ostreopsis cf. ovata. The NAAT, completed in 95 min, attained a limit of detection of 0.06 pM target synthetic DNA and was able to detect 1 ng/µL O. cf. ovata genomic DNA with negligible cross-reactivity from a closely related microalgae species. We think that the integration of thread electrodes within paper-based devices paves the way for digital one-time use NAATs and numerous other advanced electroanalytical paper- or textile-based devices.

Nitrocellulose-bound achromopeptidase for point-of-care nucleic acid tests.

Enzymes are the cornerstone of modern biotechnology. Achromopeptidase (ACP) is a well-known enzyme that hydrolyzes a number of proteins, notably proteins on the surface of Gram-positive bacteria. It is therefore used for sample preparation in nucleic acid tests. However, ACP inhibits DNA amplification which makes its integration difficult. Heat is commonly used to inactivate ACP, but it can be challenging to integrate heating into point-of-care devices. Here, we use recombinase polymerase amplification (RPA) together with ACP, and show that when ACP is immobilized on nitrocellulose paper, it retains its enzymatic function and can easily and rapidly be activated using agitation. The nitrocellulose-bound ACP does, however, not leak into the solution, preventing the need for deactivation through heat or by other means. Nitrocellulose-bound ACP thus opens new possibilities for paper-based Point-of-Care (POC) devices.

Electrochemical Detection of Genomic DNA Utilizing Recombinase Polymerase Amplification and Stem-Loop Probe.

Nucleic acid tests integrated into digital point-of-care (POC) diagnostic systems have great potential for the future of health care. However, current methods of DNA amplification and detection require bulky and expensive equipment, many steps, and long process times, which complicate their integration into POC devices. We have combined an isothermal DNA amplification method, recombinase polymerase amplification, with an electrochemical stem-loop (S-L) probe DNA detection technique. By combining these methods, we have created a system that is able to specifically amplify and detect as few as 10 copies/µL Staphylococcus epidermidis DNA with a total time to result of 70-75 min.

Microcystin LR Shows Cytotoxic Activity Against Pancreatic Cancer Cells Expressing the Membrane OATP1B1 and OATP1B3 Transporters.

Microcystin-LR (MC-LR) is a cyanobacterial cyclopeptide, known for its unique ability to cause acute liver injury. Its cellular uptake is facilitated by specific transmembrane organic anion-transporting polypeptides (OATPs) specifically OATP1B1 and 1B3. The objective of the present study was to investigate the expression of OATPs 1A2, 1B1 and 1B3 in pancreatic cancer cell lines BxPC-3 and MIA PACA-2 and assess their role in MC-LR-mediated cytotoxicity by using the novel xCELLigence system and flow cytometry. OATP1B1 and 1B3 were found to be expressed in both cell lines at both the mRNA and protein levels. The cytotoxic effects of MC-LR were proportionally related to the expression of these transporters. Moreover the cytotoxic potency of MC-LR was found superior to gemcitabine. Based on the expression of the organic anion transporting polypeptides 1B1 and 1B3 in pancreatic carcinoma tissue and cell lines and the potent cytotoxicity induced by MC-LR in vitro, we propose that this molecule could be held as structural basis for the development of novel targeted-compounds against pancreatic cancer.

Biomarkers of head and neck cancer, tools or a gordian knot?

Head and neck tumors comprise a wide spectrum of heterogeneous neoplasms for which biomarkers are needed to aid in earlier diagnosis, risk assessment and therapy response. Personalized medicine based on predictive markers linked to drug response, it is hoped, will lead to improvements in outcomes and avoidance of unnecessary treatment in carcinoma of the head and neck. Because of the heterogeneity of head and neck tumors, the integration of multiple selected markers in association with the histopathologic features is advocated for risk assessment. Validation of each biomarker in the context of clinical trials will be required before a specific marker can be incorporated into daily practice. Furthermore, we will give evidence that some proteins implicated in cell-cell interaction, such as CD44 may be involved in the multiple mechanism of the development and progression of laryngeal lesions and may help to predict the risk of transformation of the benign or precancerous lesions to cancer.

Cytokine effects on cell viability and death of prostate carcinoma cells.

We analyzed the effects of IL-13, IFN- γ , and IL-1 ß on cell viability and death of LNCaP and PC-3 cells and major signaling pathways involved in these effects. Significant increase of LNCaP cell death (apoptotic and necrotic) and increased levels of active caspase 3 were observed in cells treated with inhibitors of ERK 1/2 (UO126) and p38 (SB203580) prior to IL-1 ß treatment in comparison to cells treated with UO126, SB203580, or IL-1 ß alone. Significant increase of LNCaP but not PC-3 cell death was detected after treatment with LY-294002 (inhibitor of phosphatidylinositol 3-kinase). No significant increase of LNCaP and PC-3 cell death was observed after treatment with SP600125 (inhibitor of JNK), SB203580 (inhibitor of p38), UO126 (inhibitor of ERK 1/2), or BAY 11-7082 (inhibitor of NF- κ B). Reduced c-FLIPL expression was observed in LNCaP cells treated with LY-294002. The significant potentiation of LNCaP cell death by inhibition of ERK 1/2, p38, and PI3-K pathways may provide a rationale for therapeutic approach in androgen-dependent prostate cancer.

Cytokine effects on cell survival and death of A549 lung carcinoma cells.

PURPOSE: IL-13, TNF-α and IL-1ß have various effects on lung cancer growth and death, but the signaling pathways mediating these effects have not been extensively analyzed. Therefore, the effects of IL-13, TNF-α and IL-1ß alone, and in combination with Fas, on cell viability and death as well as major signaling pathways involved in these effects were investigated in A549 lung carcinoma cells. RESULTS: Using MTT and flow cytometry, IL-13, TNF-α and IL-1ß pretreatment decreased Fas-induced cell death. These anti-cell death effects were attenuated by pretreatment with inhibitors of Nuclear factor-κB [NF-κB], Phoshatidylinositole-3 kinase [PI3-K], JNK, p38 and ERK1/2 pathways. Using Western blot, IL-13, TNF-α and IL-1ß treated cells showed time-dependent expression of p-ERK1/2, p-p38, p-JNK, p-Akt and p-IκBα proteins, decreased IκBα protein expression, no cleavage of Caspase-3 and PARP1 proteins and no notable alterations of Fas protein. IL-13 and TNF-α treated cells showed time-dependent increase of FLIPL expression. CONCLUSION: IL-13, TNF-α and IL-1ß attenuate the pro-cell death effects of Fas on A549 cells, at least partially, by pathways involving the NF-κB, PI3-K and MAP kinases, but not by alterations of Fas protein expression. The IL-13 and TNF-α cell survival effects may also be due to increased expression of FLIPL protein.