Wastewater biofilm formation on self-assembled monolayer surfaces using elastomeric flow cells.

In anaerobic wastewater treatment, microbial biofilm is beneficial for efficient substrate utilization and for preventing the wash-out of key microorganisms. By providing solid supports, biofilm formation can be accelerated due to the early initial adhesion of residing microbes. Alteration in surface properties is therefore one such approach that helps us understand microbial interfacial interaction. Here, self-assembled monolayers of alkanethiols with carboxyl (-COOH), hydroxyl (-OH), and amine (-NH2) terminal moieties on gold (Au) substrates were employed to study the initial adhesion of wastewater microbes. An elastomeric flow cell was also utilized to simulate the environment of wastewater bioreactor. Results from fluorescence in situ hybridization (FISH) portrayed more enhanced microbial adhesion after 2 h on -NH2 functional group with the calculated surface coverage of 12.8 ±â€¯2.4% as compared to 7.7 ±â€¯1.6% on -COOH, 11.0 ±â€¯2.0% on -OH, and 1.2% on unmodified Au surfaces. This might be because of concomitant electrostatic attraction between negatively-charged bacteria and positively-charged (-NH3+) functional groups. Nevertheless, the average surface coverage by individual biofilm clusters was 28.0 ±â€¯5.0 µm2 and 32.0 ±â€¯9.0 µm2 on -NH2 and -OH surfaces, respectively, while -COOH surfaces resulted in higher value (60.0 ±â€¯5.0 µm2) and no significant cluster formation was observed on Au surfaces. Accordingly, the average inter-cluster distance observed on -NH2 surfaces was relatively smaller (3.0 ±â€¯0.6 µm) as compared to that on other surfaces. Overall, these data suggest favorable initial biofilm growth on more hydrophilic and positively-charged surfaces. Furthermore, the analysis of the mean fluorescence intensity revealed preferred initial adhesion of key microbes (archaea) on -OH and -NH2 surfaces. Indeed, results obtained from this study would be beneficial in designing selective biointerfaces for certain biofilm carriers in a typical wastewater bioreactor. Importantly, our elastomeric flow cell integrated with SAM-modified surfaces demonstrated an ideal platform for high-throughput investigation of wastewater biofilm under controlled environments.

From the bottom up: dimensional control and characterization in molecular monolayers.

Self-assembled monolayers are a unique class of nanostructured materials, with properties determined by their molecular lattice structures, as well as the interfaces with their substrates and environments. As with other nanostructured materials, defects and dimensionality play important roles in the physical, chemical, and biological properties of the monolayers. In this review, we discuss monolayer structures ranging from surfaces (two-dimensional) down to single molecules (zero-dimensional), with a focus on applications of each type of structure, and on techniques that enable characterization of monolayer physical properties down to the single-molecule scale.

Real-time kinetic analysis and detection of glycated hemoglobin A1c using a quartz crystal microbalance-based aptasensor.

Glycated hemoglobin (HbA1c) has been an important biomarker for long-term diagnosis and monitoring of diabetes mellitus. The development of a rapid, reliable, and less sophisticated device to measure HbA1c is imperative to facilitate efficient early-care diabetes management. To date, no existing aptamer-based biosensor (aptasensor) for detecting HbA1c has been developed using a quartz crystal microbalance (QCM). In this study, the aptamer specific to HbA1c as a novel biosensing receptor was covalently functionalized onto a QCM substrate via mixed self-assembled monolayers (SAMs). A portable QCM equipped with a liquid-flow module was used to investigate the biospecificity, sensitivity, and interaction dynamics of the aptamer functionalized surfaces. The real-time kinetic analysis of HbA1c binding to the surface-functionalized aptamers revealed "on" and "off" binding rates of 4.19 × 104 M-1 s-1 and 2.43 × 10-3 s-1, respectively. These kinetic parameters imply that the QCM-based aptasensor specifically recognizes HbA1c with an equilibrium dissociation constant as low as 57.99 nM. The linear detection of HbA1c spanned from 13 to 108 nM, with a limit of detection (LOD) of 26.29 nM. Moreover, the spiked plasma sample analysis offered compelling evidence that this aptasensor is a promising technique for developing a point-of-care device for diabetes mellitus.

Incident-angle-modulated molecular plasmonic switches: a case of weak exciton-plasmon coupling.

We have designed an angularly tunable plasmonic system that consists of Au nanodisks in combination with molecules of photoswitchable resonance, spiropyran, to gain new insights into weak exciton-plasmon couplings. In the weak exciton-plasmon coupling regime, switching molecular resonance can induce localized surface plasmon resonance (LSPR) peak shifts due to the change in the refractive index of the molecular materials. On the basis of the angle-resolved spectroscopic study of the nanodisk-spiropyran system both with and without UV irradiation, we reveal an unusual "zigzag" curve for the LSPR peak shifts (due to the photoswitching of the molecular resonance) as a function of the original LSPR peak wavelength. A further theoretical analysis attributes the "zigzag" curve to two significant competing effects that depend on the incident angle of the probe light: plasmon-enhanced molecular resonance absorption and LSPR sensitivity to the surroundings' refractive index.

Surface-enhanced Raman spectroscopy to probe reversibly photoswitchable azobenzene in controlled nanoscale environments.

We apply in situ surface-enhanced Raman spectroscopy (SERS) to probe the reversible photoswitching of azobenzene-functionalized molecules inserted in self-assembled monolayers that serve as controlled nanoscale environments. Nanohole arrays are fabricated in Au thin films to enable SERS measurements associated with excitation of surface plasmons. A series of SERS spectra are recorded for azobenzene upon cycling exposure to UV (365 nm) and blue (450 nm) light. Experimental spectra match theoretical calculations. On the basis of both the simulations and the experimental data analysis, SERS provides quantitative information on the reversible photoswitching of azobenzene in controlled nanoscale environments.

Reactive argon-plasma activation of screen-printed carbon electrodes for highly selective dopamine determination.

Dopamine (DA) deficiency has been linked to several psychiatric disorders. Electrochemical determination of the level of DA suffers from abundant ascorbic acid (AA) and uric acid (UA) in body fluids. In this work, a facile argon (Ar) plasma treatment was utilized to enhance the electrocatalytic reactivity of screen-printed carbon electrodes (SPCEs) for selective DA detection. Surface characterization of the Ar-treated SCPEs verified that the carbon paste binders were successfully removed and single-bonded oxygenated moieties (-OH and C-O-C) were generated. Interestingly, the sharper D* and D'' Raman interbands were new key evidence of a higher exposure of carbon defect sites. Electrochemical studies further revealed that the Ar-treated SPCEs possessed faster heterogeneous electron-transfer rates, larger electroactive surface areas, and much higher conductivity when compared with untreated electrodes. As a result, the oxidation potentials of AA, DA, and UA in the mixture could be well-resolved and the current responses were significantly increased. The selective determination of DA in the presence of AA and UA by differential pulse voltammetry gave two linear responses with the limit of detection of 0.27 µM (0.15-10 µM range). Moreover, this Ar-treated SPCE had high reproducibility and good storage stability. These results suggest that Ar-plasma treatment could be a promising method to enhance the electrocatalytic properties of SPCEs for the detection of biomolecules.

Tuning stamp surface energy for soft lithography of polar molecules to fabricate bioactive small-molecule microarrays.

Soft-lithography-based techniques are widely used to fabricate microarrays. Here, the use of microcontact insertion printing is described, a soft-lithography method specifically developed for patterning at the dilute scales necessary for highly selective biorecognition. By carefully tuning the polar surface energy of polymeric stamps, problems associated with patterning hydrophilic tether molecules inserted into hydrophilic host self-assembled monolayers (SAMs) are surmounted. Both prefunctionalized tethers and on-chip functionalization of SAMs patterned by microcontact insertion printing enable the fabrication of small-molecule microarrays. Substrates patterned with the neurotransmitter precursor 5-hydroxytryptophan selectively capture a number of different types of membrane-associated receptor proteins, which are native binding partners evolved to recognize free serotonin. These advances provide new avenues for chemically patterning small molecules and fabricating small molecule microarrays with highly specific molecular recognition capabilities.

Controlled DNA Patterning by Chemical Lift-Off Lithography: Matrix Matters.

Nucleotide arrays require controlled surface densities and minimal nucleotide-substrate interactions to enable highly specific and efficient recognition by corresponding targets. We investigated chemical lift-off lithography with hydroxyl- and oligo(ethylene glycol)-terminated alkanethiol self-assembled monolayers as a means to produce substrates optimized for tethered DNA insertion into post-lift-off regions. Residual alkanethiols in the patterned regions after lift-off lithography enabled the formation of patterned DNA monolayers that favored hybridization with target DNA. Nucleotide densities were tunable by altering surface chemistries and alkanethiol ratios prior to lift-off. Lithography-induced conformational changes in oligo(ethylene glycol)-terminated monolayers hindered nucleotide insertion but could be used to advantage via mixed monolayers or double-lift-off lithography. Compared to thiolated DNA self-assembly alone or with alkanethiol backfilling, preparation of functional nucleotide arrays by chemical lift-off lithography enables superior hybridization efficiency and tunability.

Subtractive patterning via chemical lift-off lithography.

Conventional soft-lithography methods involving the transfer of molecular "inks" from polymeric stamps to substrates often encounter micrometer-scale resolution limits due to diffusion of the transferred molecules during printing. We report a "subtractive" stamping process in which silicone rubber stamps, activated by oxygen plasma, selectively remove hydroxyl-terminated alkanethiols from self-assembled monolayers (SAMs) on gold surfaces with high pattern fidelity. The covalent interactions formed at the stamp-substrate interface are sufficiently strong to remove not only alkanethiol molecules but also gold atoms from the substrate. A variety of high-resolution patterned features were fabricated, and stamps were cleaned and reused many times without feature deterioration. The remaining SAM acted as a resist for etching exposed gold features. Monolayer backfilling into the lift-off areas enabled patterned protein capture, and 40-nanometer chemical patterns were achieved.

Native serotonin membrane receptors recognize 5-hydroxytryptophan-functionalized substrates: enabling small-molecule recognition.

Recognition of small diffusible molecules by large biomolecules is ubiquitous in biology. To investigate these interactions, it is important to be able to immobilize small ligands on substrates; however, preserving recognition by biomolecule-binding partners under these circumstances is challenging. We have developed methods to modify substrates with serotonin, a small-molecule neurotransmitter important in brain function and psychiatric disorders. To mimic soluble serotonin, we attached its amino acid precursor, 5-hydroxytryptophan, via the ancillary carboxyl group to oligo(ethylene glycol)-terminated alkanethiols self-assembled on gold. Anti-5-hydroxytryptophan antibodies recognize these substrates, demonstrating bioavailability. Interestingly, 5-hydroxytryptophan-functionalized surfaces capture membrane-associated serotonin receptors enantiospecifically. By contrast, surfaces functionalized with serotonin itself fail to bind serotonin receptors. We infer that recognition by biomolecules evolved to distinguish small-molecule ligands in solution requires tethering of the latter via ectopic moieties. Membrane proteins, which are notoriously difficult to isolate, or other binding partners can be captured for identification, mapping, expression, and other purposes using this generalizable approach.