SELF-ASSEMBLED MONOLAYERS AND METHODS FOR USING THE SAME IN BIOSENSING APPLICATIONS

Cross - linked amphiphile constructs that form self - as sembled monolayers ( SAMs ) on metal surfaces such as gold surfaces are disclosed . These new SAMs generate well packed and highly oriented monolayer films on gold sur faces . A method for using the SAMs in the fabrication of biomolecule sensors is also disclosed

A whole-cell biosensor for the detection of gold

Geochemical exploration for gold (Au) is becoming increasingly important to the mining industry. Current processes for Au analyses require sampling materials to be taken from often remote localities. Samples are then transported to a laboratory equipped with suitable analytical facilities, such as Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) or Instrumental Neutron Activation Analysis (INAA). Determining the concentration of Au in samples may take several weeks, leading to long delays in exploration campaigns. Hence, a method for the on-site analysis of Au, such as a biosensor, will greatly benefit the exploration industry. The golTSB genes from Salmonella enterica serovar typhimurium are selectively induced by Au(I/III)-complexes. In the present study, the golTSB operon with a reporter gene, lacZ, was introduced into Escherichia coli. The induction of golTSB::lacZ with Au(I/III)-complexes was tested using a colorimetric β-galactosidase and an electrochemical assay. Measurements of the β-galactosidase activity for concentrations of both Au(I)- and Au(III)-complexes ranging from 0.1 to 5 µM (equivalent to 20 to 1000 ng g⁻¹ or parts-per-billion (ppb)) were accurately quantified. When testing the ability of the biosensor to detect Au(I/III)-complexes(aq) in the presence of other metal ions (Ag(I), Cu(II), Fe(III), Ni(II), Co(II), Zn, As(III), Pb(II), Sb(III) or Bi(III)), cross-reactivity was observed, i.e. the amount of Au measured was either under- or over-estimated. To assess if the biosensor would work with natural samples, soils with different physiochemical properties were spiked with Au-complexes. Subsequently, a selective extraction using 1 M thiosulfate was applied to extract the Au. The results showed that Au could be measured in these extracts with the same accuracy as ICP-MS (P<0.05). This demonstrates that by combining selective extraction with the biosensor system the concentration of Au can be accurately measured, down to a quantification limit of 20 ppb (0.1 µM) and a detection limit of 2 ppb (0.01 µM).Carla M. Zammit, Davide Quaranta, Shane Gibson, Anita J. Zaitouna, Christine Ta, Joël Brugger, Rebecca Y. Lai, Gregor Grass, Frank Reit

Surface-based whole protein detection via electrochemical peptide-based sensors

In this work, we determined that non-covalent attachments could be used for peptide- based sensors. Similarly, different surface modifications could change the surface conformation of a peptide and even the linear dynamic range of a given peptide. Through the introduction of various types of biomolecules, as well as small organic molecules, the surface can be tuned to improve specificity and selectivity without influencing the linear dynamic range, kinetics, or binding constants of a given peptide-antibody interaction. We reported the design and characterization of a metal ion-imidazole self-assembled monolayer on a gold electrode. The resultant monolayer is well suited for direct immobilization of histidine and methylene blue-modified peptides. Multiple metal ions were used to create the peptide-imidazole interaction and multiple methods could be used to disrupt this interaction. A Ni(II)-nitrilotriacetic acid self-assembled monolayer (NTA SAM) was examined in the fabrication of an electrochemical peptide-based (E-PB) sensor for detection of anti- Ara h 2 antibodies. The performance of the sensor fabricated on a Ni(II)-NTA SAM using a His-tagged peptide was then compared with the sensor fabricated using the conventional approach via direct immobilization of a thiolated peptide. Differences occurred between the sensors\u27 detection limit. More importantly, unlike previously developed E-PB sensors, both sensors are regenerable and reusable. Short thiolated oligonucleotides were incorporated as passivating diluents in the fabrication of E-PB sensors, with the goal of creating a negatively charged layer capable of resisting non-specific adsorption of matrix contaminants. Also, small molecule amphiphiles were synthesized and incorporated as antifouling passivation diluents into an E-PB sensor. The E-PB HIV sensors fabricated using these diluents were found to be more specific and selective, while retaining attributes similar to the sensor fabricated without these diluents. We explored the use of a single peptide strand that incorporates antifouling amino acids (additional SGSGSG or EKEKEK) into an E-PB sensor biorecognition element for the detection of anti-p24 antibodies. All sensors responded only to the correct antibody in the presence of random antibodies, but we observed that sensors with the antifouling amino acids had improved performance in complex real world samples, saliva and urine

Use of thiolated oligonucleotides as anti-fouling diluents in electrochemical peptide-based sensors

We incorporated short thiolated oligonucleotides as passivating diluents in the fabrication of electrochemical peptide-based (E-PB) sensors, with the goal of creating a negatively charged layer capable of resisting nonspecific adsorption of matrix contaminants. The E-PB HIV sensors fabricated using these diluents were found to be more specific and selective, while retaining attributes similar to the sensor fabricated without these diluents. Overall, these results highlight the advantages of using oligonucleotides as anti-fouling diluents in self-assembled monolayer-based sensors

SELF-ASSEMBLED MONOLAYERS AND METHODS FOR USING THE SAME IN BIOSENSING APPLICATIONS

Cross-linked amphiphile constructs that form self-assembled monolayers (SAMs) on metal Surfaces such as gold Surfaces are disclosed. These new SAMs generate well packed and highly oriented monolayer films on gold Surfaces. A method for using the SAMs in the fabrication of biomolecule sensors is also disclosed

Selected HLA-B allotypes are resistant to inhibition or deficiency of the transporter associated with antigen processing (TAP)

<div><p>Major histocompatibility complex class I (MHC-I) molecules present antigenic peptides to CD8⁺ T cells, and are also important for natural killer (NK) cell immune surveillance against infections and cancers. MHC-I molecules are assembled via a complex assembly pathway in the endoplasmic reticulum (ER) of cells. Peptides present in the cytosol of cells are transported into the ER via the transporter associated with antigen processing (TAP). In the ER, peptides are assembled with MHC-I molecules via the peptide-loading complex (PLC). Components of the MHC-I assembly pathway are frequently targeted by viruses, in order to evade host immunity. Many viruses encode inhibitors of TAP, which is thought to be a central source of peptides for the assembly of MHC-I molecules. However, human MHC-I (HLA-I) genes are highly polymorphic, and it is conceivable that several variants can acquire peptides via TAP-independent pathways, thereby conferring resistance to pathogen-derived inhibitors of TAP. To broadly assess TAP-independent expression within the HLA-B locus, expression levels of 27 frequent HLA-B alleles were tested in cells with deficiencies in TAP. Approximately 15% of tested HLA-B allotypes are expressed at relatively high levels on the surface of TAP1 or TAP2-deficient cells and occur in partially peptide-receptive forms and Endoglycosidase H sensitive forms on the cell surface. Synergy between high peptide loading efficiency, broad specificity for peptides prevalent within unconventional sources and high intrinsic stability of the empty form allows for deviations from the conventional HLA-I assembly pathway for some HLA-B*35, HLA-B*57 and HLA-B*15 alleles. Allotypes that display higher expression in TAP-deficient cells are more resistant to viral TAP inhibitor-induced HLA-I down-modulation, and HLA-I down-modulation-induced NK cell activation. Conversely, the same allotypes are expected to mediate stronger CD8⁺ T cell responses under TAP-inhibited conditions. Thus, the degree of resistance to TAP inhibition functionally separates specific HLA-B allotypes.</p></div

RIT HLA-B allotypes are more resistant to TAP inhibition than non-RIT HLA-B allotypes.

<p>(A and B) CEM cells or (C and D) K562 cells expressing different exogenous HLA-B allotypes were infected with a HA-tagged BNLF2a-encoding retrovirus or retrovirus lacking BNLF2a (vector). BNLF2a expression levels were assessed by flow cytometry after intracellular staining with monoclonal anti-HA antibody and normalized to MFI values obtained from CEM or K562 cells lacking exogenous HLA-B (labeled vec) infected with the BNLF2a-encoding retrovirus (A and C). Cell surface HLA-B was measured by flow cytometry after staining with W6/32. The MFI ratios in cells expressing or lacking BNLF2a were calculated (BNLF2a/vector) (n = 3 measurements for each HLA-B expressing CEM cells or K562 cells from one infection with BNLF2a-encoding retrovirus) (B and D).</p

RIT HLA-B allotypes of the Bw4 group are more efficient in inhibiting KIR3DL1⁺ NK cell activation in the presence of the viral TAP inhibitor BNLF2a.

<p>HLA-I deficient K562 cells infected with retrovirus encoding exogenous HLA-B*44:03 and HLA-B*57:03 or retrovirus lacking HLA-B (vector) were chosen and further infected with a BNLF2a-encoding retrovirus or retrovirus lacking BNLF2a (vector). Intracellular BNLF2a expression levels (A) and cell surface expression of HLA-B were assessed by flow cytometry (B). HLA-B*44:03 expression is more strongly reduced by BNLF2a than HLA-B*57:03 (one representative experiment of three measurements is shown). (C) K562 cell based NK cell activation assay was performed with PBMCs from three different donors (D187, D136 and D215). CD3^-CD56⁺KIR3DL1⁺ cells were gated and NK cell activation was assessed by quantifying IFN-γ expressing population. One representative dataset from two experiments is shown.</p

TAP1-dependencies of HLA-B expression.

<p>(A) Cells from one infection from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.g001" target="_blank">Fig 1A</a> were subsequently infected with a TAP1-encoding retrovirus. The MFI ratios in the presence and absence of TAP1 (+TAP1/-TAP1) were calculated for each HLA-B–expressing cell line (n = 4 analyses from one infection). (B) Correlation between surface HLA-B expression in SK19 cells (calculated from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.g001" target="_blank">Fig 1A</a>) and their +TAP1/-TAP1 MFI ratios (calculated from Fig 2A). (C) Cells from the infections shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007171#ppat.1007171.g001" target="_blank">Fig 1D</a> were subsequently infected with a TAP1-encoding retrovirus. The MFI ratios in the presence and absence of TAP1 (+TAP1/-TAP1) were calculated for each HA-HLA-B–expressing cell line (n = 4 analyses from one infection). (D) Parental and TAP1-knockdown Hela cells (Hela-TAP1-KD) were assessed by immunoblotting with the anti-TAP1 antibody 148.3 (inset panel; 5, 10 or 20 μg of cell lysate was loaded in each lane) and infected with retroviruses encoding HLA-B allotypes or a control retrovirus (vector). TAP1 expression levels were measured by flow cytometry after intracellular staining with 148.3 antibody. (E) HLA-B expression levels at the surface of Hela or Hela-TAP1-KD cells were measured after W6/32 staining. The MFI ratios (Hela-TAP1-KD/Hela) were calculated for each HLA-B–expressing cell line (n = 6–7 measurements from three separate infections of Hela or Hela-TAP1-KD cells with retroviruses encoding indicated HLA-B). Significant differences are indicated (with an asterisk) on the graph (<i>P</i><0.05). Statistical significance is based on an ordinary one-way ANOVA analysis with Fisher’s LSD test.</p

Allotype-dependent variations in cell surface HLA-B expression in TAP-deficient cells.

<p>(A and B) Cell surface HLA-B levels in SK19 (TAP1-deficient) or STF1 (TAP2-deficient) cells infected with retroviral constructs encoding indicated HLA-B were expressed as mean fluorescence intensity (MFI) ratios relative to those obtained for infections with an empty retroviral vector lacking HLA-B (vec). Data are derived from 4–13 (A) or 2–9 (B) flow cytometric measurements with the W6/32 antibody following 2–5 (A) or 1–4 (B) separate retroviral infections. (C) The MFI ratios for HLA-B allotypes from SK19 cells (Panel A) correlate with those from STF1 cells (Panel B). (D) SK19 cells were also infected with retroviruses encoding HA-tagged versions of HLA-B (HA-HLA-B). Cell surface expression levels of HLA-B molecules were tested by flow cytometry after staining with anti-HA. Data are derived from 3 measurements following one infection. Significant differences are indicated (with an asterisk) on the graph (<i>P</i><0.05). Statistical significance is based on an ordinary one-way ANOVA analysis with Fisher’s LSD test.</p