Mass Spectrometry Imaging of Small Molecules Using Desorption/Ionization on Silicon

Development of novel tools to image spatial distribution of small molecules in biological samples is essential in disease diagnosis and biomarker discovery. To simplify sample preparation and reduce background noise in the low-mass region, we describe here the use of a matrix-free mass spectrometric imaging method, i.e., desorption/ionization on silicon (DIOS), for biological surface analysis. The imaging parameters, such as the laser beam diameter and the translation stage movement, were studied and optimized to improve imaging performance. The use of DIOS imaging to map small molecules on mouse liver tissues was demonstrated. In addition, phosphatidylcholine (PC) and propidium iodide (PI) were used as the cell membrane and nucleus markers, respectively, to “visualize” the presence of HEK 293 cells. The reconstructed ion maps of PC and PI were compared with the optical images collected from the same sample using bright-field and fluorescence microscopy. A good correlation of the spatial distribution of cells confirmed the validity of this DIOS imaging approach

Schematic representation of potential interactions between the AP-1 site and RNA pol II in the human iNOS promoter with p300 in DNA loop formation.

<p>Schematic representation of potential interactions between the AP-1 site and RNA pol II in the human iNOS promoter with p300 in DNA loop formation.</p

Effect of p300 on cytokine induced hiNOS expression.

<p>(A) Western blot of cytokine mix (CM) TNF-α + IL-1β + IFN-γ induced hiNOS protein, but not p300 nuclear proteins in human hepatocytes and A549 cells. Three similar Western blot experiments were quantified for hiNOS and p300 proteins. (B) RT-PCR analysis of hiNOS mRNA expression in human hepatocytes after overexpression of p300. Hepatocytes were transfected with p300 expression vector or control empty vector, and then treated with CM. mRNAs were extracted from hepatocytes after CM treatment for 6 hr. (C) Griess assay of NO produce in human hepatocytes. Medium from cell culture was collected from hepatocytes after CM treatment for 24 hr. The graph shows means ± SD.</p

3C assay of DNA looping.

<p>Hind III restriction enzyme spliced at -5,274 and -631 bp in the hiNOS promoter. The gel of the 3C assay shown is representative of three experiments.</p

In vitro and In vivo analysis of AP-1 binding sites in the hiNOS enhancer.

<p>(A) Mutagenesis analysis of AP-1 sites at -5.1 kb downstream (Pr8-1d) or -5.3 kb upstream (Pr8-1u) in the hiNOS promoter. Mutant construct for each site or double AP-1 mutant (1u+1d) were generated in the hiNOS Pr8 promoter luciferase reporter plasmid driven by pCMV promoter. Wild-type hiNOS promoter luciferase reporter plasmid served as control. * Indicates p <0.05 vs. p300. (B) ChIP analysis of AP-1 binding sites in the hiNOS enhancer with various antibodies. * Indicates p <0.05 vs. Ig G.</p

ChIP-Loop assay. Gel assay for AP-1, RNA pol II, and p300 binding.

<p>Schematic representation of AP-1 sites in the human iNOS promoter with relevant target sequences for Hind III restriction endonucleases and location of PCR primers. Ig G serves as negative control. Upper lane: without p300 siRNA treatment; Lower lane: with p300 siRNA treatment, p300 antibody with scrambled control siRNA serves as positive control. Gel assay shown is representative of three similar experiments.</p

p300 mediated transactivation of the hiNOS or heterologous promoter.

<p>(A) The –7.2 kb wild-type (WT) human iNOS promoter construct (Pr8) or deleted -5 to -6 kb enhancer region (Pr8-Del), were co-transfected into A549 cells with p300 expression vector. Basal and stimulated luciferase activities were determined 6 hours after cytokine mix (CM) stimulation. Relative luciferase activities (RLA) values are the means ± sd of at least three separate experiments performed in triplicate. *Indicates <i>P</i> < 0.05 <i>vs</i>. basal, # indicates P < 0.05 vs. control (B) The minimal TK promoter construct with ligated hiNOS enhancer was co-transfected into A549 cells with p300 expression vector. Co-transfection with empty vector served as control. Basal and stimulated luciferase activity was determined 6 hr after cytokine stimulation. Values shown are the means ± sd of at least three separate experiments performed in triplicate. *Indicates P < 0.05 vs. basal, # indicates P < 0.05 vs. control.</p

Nanostructured Interface Loaded with Chimeric Enzymes for Fluorimetric Quantification of Cyclosporine A and FK506

Advances in protein engineering resulted in increased efforts to create protein biosensors that can replace instrumentation-heavy analytical and diagnostic methods. Sensitivity, amenability to multiplexing, and manufacturability remain to be among the key issues preventing broad utilization of protein biosensors. Here, we attempt to address these by constructing arrays utilizing protein biosensors based on the artificial allosteric variant of PQQ-glucose dehydrogenase (GDH). We demonstrated that the silica nanoparticle-immobilized GDH protein could be deposited on fiberglass sheets without loss of activity. The particle-associated GDH activity could be monitored using changes in the fluorescence of the commonly used electron mediator phenazine methosulfate. The constructed biosensor arrays of macrocyclic immunosuppressant drugs cyclosporine A and FK-506 displayed very low background and a remarkable dynamic range exceeding 300-fold that resulted in a limit of detection of 2 pM for both analytes. This enabled us to quantify both drugs in human blood, serum, urine, and saliva. The arrays could be stored in dry form and quantitatively imaged using a smartphone camera, demonstrating the method’s suitability for field and point-of-care applications. The developed approach provides a generalizable platform for biosensor array development that is compatible with inexpensive and potentially scalable manufacturing

Blood Compatibility Evaluations of Fluorescent Carbon Dots

Because of their unique advantages, fluorescent carbon dots are gaining popularity in various biomedical applications. For these applications, good biosafety is a prerequisite for their use in vivo. Studies have reported the preliminary biocompatibility evaluations of fluorescent carbon dots (mainly cytotoxicity); however, to date, little information is available about their hemocompatibility, which could impede their development from laboratory to bedside. In this work, we evaluated the hemocompatibility of fluorescent carbon dots, which we prepared by hydrothermal carbonization of α-cyclodextrin. The effects of the carbon dots on the structure and function of key blood components were investigated at cellular and molecular levels. In particular, we considered the morphology and lysis of human red blood cells, the structure and conformation of the plasma protein fibrinogen, the complement activation, platelet activation, and in vitro and in vivo blood coagulation. We found that the carbon dots have obvious concentration-dependent effects on the blood components. Overall, concentrations of the fluorescent carbon dots at ≤0.1 mg/mL had few adverse effects on the blood components, but at higher doses, the carbon dots impair the structure and function of the blood components, causing morphological disruptions and lysis of red blood cells, interference in the local microenvironments of fibrinogen, activation of the complement system, and disturbances in the plasma and whole blood coagulation function in vitro. However, the carbon dots tend to activate platelets only at low concentrations. Intravenous administration of the carbon dots at doses up to 50 mg/kg did not impair the blood coagulation function. These results provide valuable information for the clinical application of fluorescent carbon dots

Engineered PQQ-Glucose Dehydrogenase as a Universal Biosensor Platform

Biosensors with direct electron output hold promise for nearly seamless integration with portable electronic devices. However, so far, they have been based on naturally occurring enzymes that significantly limit the spectrum of detectable analytes. Here, we present a novel biosensor architecture based on analyte-driven intermolecular recombination and activity reconstitution of a re-engineered component of glucometers: PQQ-glucose dehydrogenase. We demonstrate that this sensor architecture can be rapidly adopted for the detection of immunosuppressant drugs, α-amylase protein, or protease activity of thrombin and Factor Xa. The biosensors could be stored in dried form without appreciable loss of activity. We further show that ligand-induced activity of the developed biosensors could be directly monitored by chronoamperometry, enabling construction of disposable sensory electrodes. We expect that this architecture could be expanded to the detection of other biochemical activities, post-translational modifications, nucleic acids, and inorganic molecules