Staphylococcus aureus Detection in Milk Using a Thickness Shear Mode Acoustic Aptasensor with an Antifouling Probe Linker.

Contamination of food by pathogens can pose a serious risk to health. Therefore, monitoring for the presence of pathogens is critical to identify and regulate microbiological contamination of food. In this work, an aptasensor based on a thickness shear mode acoustic method (TSM) with dissipation monitoring was developed to detect and quantify Staphylococcus aureus directly in whole UHT cow's milk. The frequency variation and dissipation data demonstrated the correct immobilization of the components. The analysis of viscoelastic properties suggests that DNA aptamers bind to the surface in a non-dense manner, which favors the binding with bacteria. The aptasensor demonstrated high sensitivity and was able to detect S. aureus in milk with a 33 CFU/mL limit of detection. Analysis was successful in milk due to the sensor's antifouling properties, which is based on 3-dithiothreitol propanoic acid (DTTCOOH) antifouling thiol linker. Compared to bare and modified (dithiothreitol (DTT), 11-mercaptoundecanoic acid (MUA), and 1-undecanethiol (UDT)) quartz crystals, the sensitivity of the sensor's antifouling in milk improved by about 82-96%. The excellent sensitivity and ability to detect and quantify S. aureus in whole UHT cow's milk demonstrates that the system is applicable for rapid and efficient analysis of milk safety.

Long-Term Reduction of Bacterial Adhesion on Polyurethane by an Ultra-Thin Surface Modifier.

Indwelling urinary catheters are employed widely to relieve urinary retention in patients. A common side effect of the use of these catheters is the formation of urinary tract infections (UTIs), which can lead not only to severe medical complications, but even to death. A number of approaches have been used to attempt reduction in the rate of UTI development in catheterized patients, which include the application of antibiotics and modification of the device surface by coatings. Many of these coatings have not seen use on catheters in medical settings due to either the high cost of their implementation, their long-term stability, or their safety. In previous work, it has been established that the simple, stable, and easily applicable sterilization surface coating 2-(3-trichlorosilylpropyloxy)-ethyl hydroxide (MEG-OH) can be applied to polyurethane plastic, where it greatly reduces microbial fouling from a variety of species for a 1-day time period. In the present work, we establish that this coating is able to remain stable and provide a similarly large reduction in fouling against Escherichia coli and Staphylococcus aureus for time periods in an excess of 30 days. This non-specific coating functioned against both Gram-positive and Gram-negative bacteria, providing a log 1.1 to log 1.9 reduction, depending on the species and day. This stability and continued efficacy greatly suggest that MEG-OH may be capable of providing a solution to the UTI issue which occurs with urinary catheters.

Detection of E. coli Bacteria in Milk by an Acoustic Wave Aptasensor with an Anti-Fouling Coating.

Milk is a significant foodstuff around the world, being produced and consumed in large quantities. The safe consumption of milk requires that the liquid has an acceptably low level of microbial contamination and has not been subjected to spoiling. Bacterial safety limits in milk vary by country but are typically in the thousands per mL of sample. To rapidly determine if samples contain an unsafe level of bacteria, an aptamer-based sensor specific to Escherichia coli bacteria was developed. The sensor is based on an ultra-high frequency electromagnetic piezoelectric acoustic sensor device (EMPAS), with the aptamer being covalently bound to the sensor surface by the anti-fouling linker, MEG-Cl. The sensor is capable of the selective measurement of E. coli in PBS and in cow's milk samples down to limits of detection of 35 and 8 CFU/mL, respectively, which is well below the safe limits for commercial milk products. This sensing system shows great promise for the milk industry for the purpose of rapid verification of product safety.

Interaction of Lipopolysaccharide-Spiked Blood with Anti-Fouling Polymyxin B-Modified Glass.

Bacterial endotoxin, also known as lipopolysaccharide (LPS), plays a major role in the initiation of sepsis, a severe inflammatory condition. Removal of the toxin from blood is one accepted method of patient treatment. Polymyxin B (PMB)-modified columns have been employed successfully for this purpose via extra-corporeal blood-flow systems that incorporate a cartridge for toxin removal. Herein we demonstrate that PMB-modified glass beads are able to reduce the presence of LPS competitively with the equivalent fiber column used in a commercial cartridge. Analysis by gas chromatography-mass spectrometry and ELISA of released fatty acids from the toxin indicates that PMB does not physically capture or significantly remove LPS from the blood samples. In reality, interaction between the surface-bound PMB and the toxin may lead to disaggregation or monomerization of LPS aggregates. As aggregates are the bioactive form of LPS, it is possible that the monomerization of these entities may be the mechanism by which their toxicity is reduced. Moreover, this work indicates that LPS monomers are stabilized subsequent to disaggregation induced by PMB.

Surface Adsorption of the Cancer Biomarker Lysophosphatidic Acid in Serum Studied by Acoustic Wave Biosensor.

The thickness shear mode acoustic wave device is of interest for the sensing of biomarkers for diseases in various biological fluids, but suffers from the issue of non-specific adsorption of compounds other than those of interest to the electrode surface, thus affecting the device's output. The aim of this present study was to determine the level of non-specific adsorption on gold electrodes from serum samples with added ovarian cancer biomarker lysophosphatidic acid in the presence of a surface anti-fouling layer. The latter was an oligoethylene molecule with thiol group for attachment to the electrode surface. It was found that the anti-fouling layer had a minimal effect on the level of both adsorption of components from serum and the marker. This result stands in sharp contrast to the analogous monolayer employed for anti-fouling reduction on silica.

Anti-Thrombogenicity Study of a Covalently-Attached Monolayer on Stent-Grade Stainless Steel.

Implantable devices fabricated from austenitic type 316L stainless steel have been employed significantly in medicine, principally because the material displays excellent mechanical characteristics and corrosion resistance. It is well known, however, that interaction of exposure of such a material to blood can initiate platelet adhesion and blood coagulation, leading to a harmful medical condition. In order to prevent undesirable surface platelet adhesion on biomaterials employed in procedures such as renal dialysis, we developed an ultrathin anti-thrombogenic covalently attached monolayer based on monoethylene glycol silane chemistry. This functions by forming an interstitial hydration layer which displays restricted mobility in the prevention of surface fouling. In the present work, the promising anti-thrombogenic properties of this film are examined with respect to platelet aggregation on 316L austenitic stainless steel exposed to whole human blood. Prior to exposure with blood, all major surface modification steps were examined by X-ray photoelectron spectroscopic analysis and surface free-angle measurement by contact angle goniometry. End-stage anti-thrombogenicity detection after 20 min of blood exposure at 100 s-1, 300 s-1, 600 s-1, 750 s-1, and 900 s-1 shear rates revealed that a significant reduction (>90%) of platelet adhesion and aggregation was achieved for surface-modified steel, compared with untreated material. This result is confirmed by experiments conducted in real time for 60-minute exposure to blood at 100 s-1, 600 s-1, and 900 s-1 shear rates.

Assembling Surface Linker Chemistry with Minimization of Non-Specific Adsorption on Biosensor Materials.

The operation of biosensors requires surfaces that are both highly specific towards the target analyte and that are minimally subject to fouling by species present in a biological fluid. In this work, we further examined the thiosulfonate-based linker in order to construct robust and durable self-assembling monolayers (SAMs) onto hydroxylated surfaces such as silica. These SAMs are capable of the chemoselective immobilization of thiol-containing probes (for analytes) under aqueous conditions in a single, straightforward, reliable, and coupling-free manner. The efficacy of the method was assessed through implementation as a biosensing interface for an ultra-high frequency acoustic wave device dedicated to the detection of avidin via attached biotin. Fouling was assessed via introduction of interfering bovine serum albumin (BSA), IgG antibody, or goat serum. Improvements were investigated systematically through the incorporation of an oligoethylene glycol backbone employed together with a self-assembling diluent without a functional distal group. This work demonstrates that the incorporation of a diluent of relatively short length is crucial for the reduction of fouling. Included in this work is a comparison of the surface attachment of the linker to Si3N4 and AlN, both materials used in sensor technology.

Reduction of microbial adhesion on polyurethane by a sub-nanometer covalently-attached surface modifier.

Indwelling urinary catheters are a common medical device used to relieve urinary retention. Many patients who undergo urinary catheterization develop urinary tract infections (UTIs), which can lead to severe medical complications and high cost of subsequent treatment. Recent years have seen a number of attempts at reducing the rate of UTIs in catheterized patients via catheter surface modifications. In this work, a low cost, robust anti-thrombogenic, and sterilizable anti-fouling layer based on a covalently-bound monoethylene glycol hydroxide (MEG-OH) was attached to polyurethane, a polymeric material commonly used to fabricate catheters. Modified polyurethane tubing was compared to bare tubing after exposure to a wide spectrum of pathogens including Gram-negative bacteria (Pesudomonas aeruginosa, Escherichia coli), Gram-positive bacteria (Staphylococcus aureus) and a fungus (Candida albicans). It has been demonstrated that the MEG-OH monolayer was able to significantly reduce the amount of adhesion of pathogens present on the material surface, with between 85 and 96 % reduction after 24 h of exposure. Additionally, similar reductions in surface fouling were observed following autoclave sterilization, long term storage of samples in air, and longer exposure up to 3 days.

Detection of the Ovarian Cancer Biomarker Lysophosphatidic Acid in Serum.

Lysophosphatidic acid (LPA) is present during the medical condition of ovarian cancer at all stages of the disease, and, therefore possesses considerable potential as a biomarker for screening its presence in female patients. Unfortunately, there is currently no clinically employable assay for this biomarker. In the present work, we introduce a test based on the duel protein system of actin and gelsolin that could allow the quantitative measurement of LPA in serum samples in a biosensing format. In order to evaluate this possibility, actin protein was dye-modified and complexed with gelsolin protein, followed by surface deposition onto silica nanoparticles. This solid-phase system was exposed to serum samples containing various concentrations of LPA and analyzed by fluorescence microscopy. Measurements conducted for the LPA-containing serum samples were higher after exposure to the developed test than samples without LPA. Early results suggest a limit of detection of 5 µM LPA in serum. The eventual goal is to employ the chemistry described here in a biosensor configuration for the large population-scale, rapid screening of women for the potential occurrence of ovarian cancer.