Rapid and dynamic detection of antimicrobial treatment response using spectral amplitude modulation in MZO nanostructure-modified quartz crystal microbalance.

We report a dynamic and rapid detection of the response of S. epidermidis to various antimicrobial treatments utilizing the real-time spectral amplitude modulations of the magnesium zinc oxide nanostructure-modified quartz crystal microbalance (MZOnano-QCM) biosensor. The sensor consists of a quartz crystal microbalance (QCM) with magnesium zinc oxide (MZO) nanostructures grown directly on the sensing electrode using metalorganic chemical vapor deposition (MOCVD). Combining the high sensitivity detection of bacteria provided by the MZO nanostructures with the QCM's dynamic acoustic spectrum makes a highly-sensitive dynamic biosensor well-suited for monitoring viscoelastic transitions during drug treatment compared to the QCM's conventional frequency shift signals. We demonstrated dynamically monitoring the response of S. epidermidis to various concentrations of the drug ciprofloxacin, and response to three different antimicrobials vancomycin, oxacillin, and ciprofloxacin, using spectral amplitude modulations of the MZOnano-QCM. Our results indicate that the amplitude modulations exhibit high sensitivity to S. epidermidis response to different drug treatments compared to the conventional frequency shift signals of the device, allowing for rapid determination (within 1.5 h) of the efficacy of the antimicrobial drug. The high sensitivity demonstrated by the spectral amplitude modulations is attributed to the direct relationship of these signals to the viscoelastic transitions of the bacterial cells on the device's sensing area while responding to drug treatment. This relationship is established by the Butterworth-Van-Dyke (BVD) model of the MZOnano-QCM. Standard microbiological protocols and assays were performed to determine the optimal drug dosages and the minimum inhibitory concentrations to serve as the benchmark for the sensor data.

Early stage detection of Staphylococcus epidermidis biofilm formation using MgZnO dual-gate TFT biosensor.

Early stage detection of biofilm formation is an important aspect of microbial research because once formed, biofilms show serious tolerance to antibiotics in contrast to the free-floating bacteria, which significantly increases the difficulty for clinical treatment of bacterial infections. The early stage detection technology is desired to improve the efficiency of medical treatments. In this work, we present a biosensor consisting of a magnesium zinc oxide (MZO) dual gate thin-film transistor (DGTFT) as the actuator and an MZO nanostructure (MZOnano) array coated conducting pad as the extended sensing gate for the early stage detection of Staphylococcus epidermidis (S. epidermidis) biofilm formation. S. epidermidis bacteria were cultured in vitro on the nanostructure modified sensing pad. Charge transfer occurs between microbial cells and the MZOnano during the initial bacterial adhesion stage. Such electrical signals, which represent the onset of biofilm formation, were dynamically detected by the DGTFT where the top gate electrode was connected to the extended MZOnano sensing pad and the bottom gate was used for biasing the device into the optimum characteristic region for high sensitivity and stable operation. The testing results show that a current change of ~80% is achieved after ~200 min of bacterial culturing. A crystal violet staining-based assay shows that tiny bacterial microcolonies just start to form at 200 min, and that it would take approximately 24 h to form matured biofilms. This technology enables medical professionals to act promptly on bacterial infection before biofilms get fully established.

Functionalization of MgZnO nanorod films and characterization by FTIR microscopic imaging.

Metal organic chemical vapor deposition grown films consisting of MgxZn1-xO (4% < x < 5%) nanorod arrays (MgZnOnano) were functionalized with 11-azidoundecanoic acid (1). The MgZnOnano was used instead of pure ZnO to take advantage of the etching resistance of the MgZnOnano during the binding and subsequent sensing device fabrication processes of sensor devices, while the low Mg composition level ensures that selected ZnO properties useful for sensors development, such as piezoelectricity, are retained. Compound 1 was bound to the MgZnOnano surface through the carboxylic acid group, leaving the azido group available for click chemistry and as a convenient infrared spectroscopy (IR) probe. The progress of the functionalization with 1 was characterized by FTIR microscopic imaging as a function of binding time, solvents employed, and MgZnOnano morphology. Binding of 1 was most stable in solutions of 3-methoxypropionitrile (MPN), a non-protic polar solvent. This occurred first in µm-scale islands, then expanded to form a rather uniform layer after 22 h. Binding in alcohols resulted in less homogenous coverage, but the 1/MgZnOnano films prepared from MPN were stable upon treatment with alcohols at room temperature. The binding behavior was significantly dependent on the surface morphology of MgZnOnano. Graphical abstract The functionalization of MgZnO nanorod films with a click-ready linker and its dependence on bidning conditions and morphology has been studied by FTIR microscopic imaging using the azido group as the IR tag.

Dynamic monitoring of antimicrobial resistance using magnesium zinc oxide nanostructure-modified quartz crystal microbalance.

Antimicrobial resistance (AMR) is becoming a major global-health concern prompting an urgent need for highly-sensitive and rapid diagnostic technology. Traditional assays available for monitoring bacterial cultures are time-consuming and labor-intensive. We present a magnesium zinc oxide (MZO) nanostructure-modified quartz crystal microbalance (MZOnano-QCM) biosensor to dynamically monitor antimicrobial effects on E. coli and S. cerevisiae. MZO nanostructures were grown on the top electrode of a standard QCM using metal-organic chemical-vapor deposition (MOCVD). The MZO nanostructures are chosen for their multifunctionality, biocompatibility, and giant effective sensing surface. The MZO surface-wettability and morphology are controlled, offering high-sensitivity to various biological/biochemical species. MZO-nanostructures showed over 4-times greater cell viability over ZnO due to MZO releasing 4-times lower Zn2+ density in the cell medium than ZnO. The MZOnano-QCM was applied to detect the effects of ampicillin and tetracycline on sensitive and resistant strains of E.coli, as well as effects of amphotericin-B and miconazole on S. cerevisiae through the device's time-dependent frequency shift and motional resistance. The MZOnano-QCM showed 4-times more sensitivity over ZnOnano-QCM and over 10-times better than regular QCM. For comparison, the optical density at 600nm (OD600) method and the cell viability assay were employed as standard references to verify the detection results from MZOnano-QCM. In the case of S. cerevisiae, the OD600 method failed to distinguish between cytotoxic and cytostatic drug effects whereas the MZOnano-QCM was able to accurately detect the drug effects. The MZOnano-QCM biosensor provides a promising technology enabling dynamic and rapid diagnostics for antimicrobial drug development and AMR detection.

Functionalization of nanostructured ZnO films by copper-free click reaction.

The copper-free click reaction was explored as a surface functionalization methodology for ZnO nanorod films grown by metal organic chemical vapor deposition (MOCVD). 11-Azidodecanoic acid was bound to ZnO nanorod films through the carboxylic acid moiety, leaving the azide group available for Cu-free click reaction with alkynes. The azide-functionalized layer was reacted with 1-ethynylpyrene, a fluorescent probe, and with alkynated biotin, a small biomolecule. The immobilization of pyrene on the surface was probed by fluorescence spectroscopy, and the immobilization of biotin was confirmed by binding with streptavidin-fluorescein isothiocyanate (streptavidin-FITC). The functionalized ZnO films were characterized by Fourier transform infrared attenuated total reflectance (FTIR-ATR), steady-state fluorescence emission, fluorescence microscopy, and field emission scanning electron microscopy (FESEM).

ZnO nanostructure-modified QCM for dynamic monitoring of cell adhesion and proliferation.

Noninvasive examination of live cell function in real-time is essential in advancing the understanding of the dynamic progression of cell's biological processes. We present a dynamic and noninvasive method of monitoring the adhesion and proliferation of bovine aortic endothelial cells (BAEC) using a ZnO nanostructure-modified quartz crystal microbalance (ZnOnano-QCM) biosensor. The ZnOnano-QCM biosensor consists of a conventional QCM with ZnO nanostructures directly grown on its sensing electrode deployed in-situ of a standard cell culture environment. Cell adhesion to the ZnO surfaces with various morphologies is studied and the optimal morphology is chosen for the BAEC adhesion. The ZnOnano-QCM biosensor displays enhanced sensitivity compared to the standard QCM sensor with ~10 times higher frequency shift and motional inductance, and ~4 times higher measured motional resistance at full confluency. The dynamic motional resistance and inductance relating to the cells' viscoelastic properties during growth are extracted from the measured time-evolving acoustic spectra. The Butterworth-Van-Dyck (BVD) model is adapted for the ZnOnano-QCM biosensor system and is used to correlate the measured time-evolving acoustic spectra with the motional characteristics of cell attachment and proliferation.

Label-free polypeptide-based enzyme detection using a graphene-nanoparticle hybrid sensor.

A graphene-nanoparticle (NP) hybrid biosensor that utilizes an electrical hysteresis change to detect the enzymatic activity and concentration of Carboxypeptidase B was developed. The results indicate that the novel graphene-NP hybrid biosensor, utilizing electrical hysteresis, has the ability to detect concentrations of targeted enzyme on the micromolar scale. Furthermore, to the knowledge of the authors, this is the first demonstration of a graphene-based biosensor that utilizes a hysteresis change resulting from metallic NPs assembled on a graphene surface.

Morphology effects on the biofunctionalization of nanostructured ZnO.

A stepwise surface functionalization methodology was applied to nanostructured ZnO films grown by metal organic chemical vapor deposition (MOCVD) having three different surface morphologies (i.e., nanorod layers (ZnO films-N), rough surface films (ZnO films-R), and planar surface films (ZnO films-P). The films were grown on glass substrates and on the sensing area of a quartz crystal microbalance (nano-QCM). 16-(2-Pyridyldithiol)-hexadecanoic acid (PDHA) was bound to ZnO films-N, -R, and -P through the carboxylic acid unit, followed by a nucleophilic displacement of the 2-pyridyldithiol moiety by single-stranded DNA capped with a thiol group (SH-ssDNA). The resulting ssDNA-functionalized films were hybridized with complementary ssDNA tagged with fluorescein (ssDNA-Fl). In a selectivity control experiment, no hybridization occurred upon treatment with non complementary DNA. The ZnO films' surface functionalization, characterized by FT-IR-ATR and fluorescence spectroscopy and detected on the nano-QCM, was successful on films-N and -R but was barely detectable on the planar surface of films-P.

Stepwise functionalization of ZnO nanotips with DNA.

A surface functionalization methodology for the development of ZnO nanotips biosensors that can be integrated with microelectronics was developed. Two types of long chain carboxylic acids linkers were employed for the functionalization of 0.5 mum thick MOCVD-grown ZnO nanotip films with single-stranded DNA (ssDNA), followed by hybridization with complementary ssDNA tagged with fluorescein. The ZnO functionalization strategy was developed for the fabrication of ZnO nanotips-linker-biomolecule films integrated with bulk acoustic wave (BAW) biosensors, and it involved three main steps. First, 16-(2-pyridyldithiol)hexadecanoic acid or N-(15-carboxypentadecanoyloxy)succinimide, both bifunctional C16 carboxylic acids, were bound to ZnO nanotip films through the COOH group, leaving at the opposite end of the alkyl chain a thiol group protected as a 2-pyridyl disulfide, or a carboxylic group protected as a N-succinimide, respectively. In the second step, ssDNA was covalently linked to each type of ZnO-linker film: the 2-pyridyl disulfide end group was substituted with 16 bases 5'-thiol-modified DNA (SH-ssDNA), and the N-succinimide ester end group was substituted with 16 bases 5'-amino-modified DNA (NH(2)-ssDNA). In the third step, the DNA-functionalized ZnO nanotip films were hybridized with complementary 5'-fluorescein ssDNA. The surface-modified ZnO nanotip films were characterized after each step by FT-IR-ATR, fluorescence emission spectroscopy, and fluorescence microscopy. This functionalization approach allows sequential reactions on the surface and, in principle, can be extended to numerous other molecules and biomolecules.