Nonspecific Adsorption Research Articles

Unraveling the interplay of DNAzyme and interfacial factors for enhanced biosensing

Deoxyribozyme (DNAzyme) and its substrate hybridization are crucial for achieving desirable detection performance in the DNAzyme coupling nanomaterial biosensor system. However, interfacial factors such as electrostatic repulsion, steric hindrance, and nonspecific adsorption from gold nanoparticles make this hybridization process complicated and challenging. Moreover, the DNAzyme structure changes with different application purposes, which might affect the DNAzyme and substrate's connection. Few studies have focused on the interplay of DNAzyme and interfacial factors in the biosensor field. In this work, three types of DNAzyme variants were designed, and their biosensor performance rules were studied and summarized with the synergistic effect of interfacial factors. Additionally, corresponding biosensor applications, such as multiple modulation functions and miRNA detections, were constructed based on the distinct principles of DNAzyme variants.

Development of target-based cell membrane affinity ultrafiltration technology for a simplified approach to discovering potential bioactive compounds in natural products.

Target-based drug discovery technology based on cell membrane targets has gained significant traction and has been steadily advancing. However, current methods still face certain limitations that need to be addressed. One of the challenges is the laborious preparation process of screening materials, which can be time-consuming and resource-intensive. Additionally, there is a potential issue of non-specific adsorption caused by carrier materials, which can result in false-positive results and compromise the accuracy of the screening process. To address these challenges, this paper proposes a target-based cell membrane affinity ultrafiltration technology for active ingredient discovery in natural products. In this technique, the cell membranes of human lung adenocarcinoma epithelial cells (A549) with a high expression of epidermal growth factor receptor (EGFR) were incubated with candidate drugs and then transferred to an ultrafiltration tube. Through centrifugation, components that interacted with EGFR were retained in the ultrafiltration tube as "EGFR-ligand" complex, while the components that did not interact with EGFR were separated. After thorough washing and eluting, the components interacting with EGFR were dissociated and further identified using LC-MS, enabling the discovery of bioactive compounds. Moreover, the target-based cell membrane affinity ultrafiltration technology exhibited commendable binding capacity and selectivity. Ultimately, this technology successfully screened and identified two major components from the Curcumae Rhizoma-Sparganii Rhizoma (CS) herb pair extracts, which were further validated for their potential anti-tumor activity through pharmacological experiments. By eliminating the need for laborious preparation of screening materials and the potential non-specific adsorption caused by carriers, the development of target-based cell membrane affinity ultrafiltration technology provides a simplified approach and method for bioactive compounds discovery in natural sources.

In Situ Spatiotemporal SERS Profiling of Bacterial Quorum Sensing by Hierarchical Hydrophobic Plasmonic Arrays in Agar Medium.

A feedback inhibition effect of high autoinducer levels on metabolite secretion in Chromobacterium subtsugae (C. subtsugae) was evidenced by in situ spatiotemporal surface-enhanced Raman spectroscopy (SERS) profiling. The hierarchical hydrophobic plasmonic array in agar medium is structured by oil/water/oil (OL/W/OH) triphasic interfacial self-assembly. The hydrophobic layer acts as a "door curtain" to selectively permit adsorption of a quorum sensing (QS)-regulated fat-soluble metabolite, i.e., violacein (Vio), and significantly blocks nonspecific adsorption of water-soluble proteins, etc. The SERS profiling clearly evidences that the diffusion of N-hexanoyl-l-homoserine lactone (C6-HSL) in agar medium quickly triggers the initial synthesis of Vio in C. subtsugae CV026 but surprisingly inhibits the intrinsic synthesis of Vio in C. subtsugae ATCC31532. The latter negative response might be related to the VioS repressor of ATCC31532, which negatively controls violacein production without influencing the expression of the CviI/R QS system. Moreover, two sender-receiver systems are constructed by separately coculturing CV026 or ATCC31532 with Hafnia alvei H4 that secretes large amounts of C6-HSL. Expectedly, the cocultivation similarly triggers the initial synthesis of Vio in CV026 but seems to have a quite weak negative effect on the intrinsic synthesis in ATCC31532. In fact, the negative regulation in ATCC31532 might be affected by a diffusion-dependent concentration effect. The H4 growth and its secretion of C6-HSL are a slow and continuous process, thereby avoiding the gathering of local high concentrations. Overall, our study put forward an in situ SERS strategy as an alternative to traditional bioluminescent tools for highly sensitively analyzing the spatiotemporal communication and cooperation in live microbial colonies.

One-step prepared of e-Gelatin-CNTs nano-composite fiber modified carbon fiber microelectrode for continuous dopamine monitoring: Characterization, biocompatibility evaluation and in vivo experiment

Fabricating electrochemical sensor capable of stable sensing in complex organism's environment will undoubtedly underpin key in the development of implanted electrochemical sensor. However, nonspecific protein adsorption can greatly reduce sensing sensitivity of electrode due to the abundance of protein in vivo. Herein, we aim to fabricating a modified layer of good protein adsorption resistance. The nano-composite fibers from gelatin with good hydrophily and multiwall carbon nanotubes (CNTs) for constructing on the surface of carbon fiber microelectrode using one-step electrospinning technology (e-Gelatin-CNTs/CFME). A comprehensive discussing on the protein adsorption resistance of e-Gelatin-CNTs/CFME that are, through the use of SEM, AFM, water contact angle, UV–vis, cyclic voltammetry and I-T. The resulting electrochemical sensor revealed stable sensing sensitivity in protein solution, a slightly decrease (0.02nA/μM). Furthermore, the sensor was successfully used for the detection of DA in human blood. More remarkable, e-Gelatin-CNTs nano-composite fibers presented excellent biocompatibility, which could meet the biosafety as implantation materials in vivo. This work provided a platform for the development of implantable electrochemical sensor.

Extraction-Less Amperometric Detection of Tetrodotoxin in Seawater

Tetrodotoxin (TTX), a potent neurotoxin, has recently been detected in marine fauna and waters in multiple locations outside its historical range and is projected to increase in both stability and bioavailability in acidifying oceans. We report the first biosensor designed to detect sub-ng ml−1 concentrations of TTX directly in natural seawater. The compact sensor chip employs a competitive enzyme strategy with a glucose oxidase-TTX conjugate that generates an amperometric response in the presence of a ferricyanide redox mediator. The biorecognition layer synthesis is confirmed by attenuated total reflection Fourier transform infrared spectroscopy. Cyclic voltammetry and electrochemical impedance spectroscopy are used to probe the stability and hydrophilicity of the biorecognition layer. We investigate the effects of nonspecific adsorption on sensor results and find that bovine serum albumen is an excellent blocking agent for preventing NSA when incubating with filtered seawater samples. The biosensor has a limit of detection of 0.053 ng ml−1 and a sensitivity of 87 nA ng−1 ml. Requiring no extraction or preconcentration steps with a time-to-results of under an hour, our biosensor provides a new tool for detecting TTX in natural waters, which can lead to more proactive monitoring and higher throughput testing.

Enhanced Adsorption Stability and Biofunction Durability with Phosphonate-Grafted, PEGylated Copolymer on Hydroxyapatite Surface.

Nonfouling surfaces are crucial in applications such as biosensors, medical implants, marine coatings, and drug delivery vehicles. However, their long-term coating stability and robust surface binding strength in physiological media remain challenging. Herein, a phosphonate-grafted, PEGylated copolymer on the hydroxyapatite (HA) surface is proposed to significantly improve the adsorption stability and thus enhance the biofunction durability accordingly. The phosphoryl (-PO3) grafted branch is employed in the functional polymer to facilitate attaching to the HA substrate. In addition, the polymer integrates the nonfouling polymer brushes of poly(ethylene glycol) (PEG) with the cell-adhesive moiety of cyclic Arg-Gly-Asp-d-Phe-Cys peptides (cRGD). A systematic study on the as-synthesized PEGylated graft copolymer indicates a synergistic binding mechanism of the NH2 and PO3 groups to HA, achieving a high surface coverage with desirable adsorption stability. The cRGD/PEGylated copolymers of optimized grafting architecture are proven to effectively adsorb to HA surfaces as a self-assembled copolymer monolayer, showing stability with minimal desorption even in a complex, physiological medium and effectively preventing nonspecific protein adsorption as examined with X-ray photoelectron spectroscopy (XPS) and a quartz crystal microbalance with dissipation (QCM-D). Direct adhesion assays further confirm that the enhanced coating stability and biofunction durability of the phosphonate-grafted, cRGD-PEGylated copolymer can considerably promote osteoblast attachment on HA surfaces, meanwhile preventing microbial adhesion. This research has resulted in a solution of self-assembly polymer structure optimization that exhibits stable nonfouling characteristics.

θ-Nanopore Ratiometry.

Developing nanoscale ratiometric techniques capable of biochemical response should prove of significance for precise applications with stringent spatial and biological restrictions. Here we present and devise the concept of θ-nanopore ratiometry, which uses ratiometric signals that could well address the serious concerns about device deviation in fabrication and nonspecific adsorption in the detection. As exemplified by a 200 nm θ-nanopore toward miRNA detection, the ±20 nm aperture drift could be mitigated and the issue of nonspecific adsorption could be minimized in the complex cytosolic environment. Practical application of this θ-nanopore ratiometry realizes the measurements of cytosolic miRNA-10b. This work has not only established a nanoscopic ratiometric technique but also enriched the extant armory of nanotools for single-cell studies and beyond.

Innovative approaches to suppress non-specific adsorption in molecularly imprinted polymers for sensing applications

Molecularly imprinted polymers (MIPs) are synthetic antibodies developed to bind selectively with specific molecules. They function through a particular recognition process involving their cavities and functional groups. Nevertheless, functional groups located outside these cavities are the main cause of non-specific molecule binding, thus reducing the effectiveness of MIPs in sensing applications. This work focused on enhancing the selectivity and performance of MIPs through electrostatic modification with surfactants. The study investigates the use of two surfactants, namely sodium dodecyl sulfate (SDS) and cetyl trimethyl ammonium bromide (CTAB), to eliminate non-specific adsorption in MIPs. The binding isotherms of the target molecule sulfamethoxazole (SMX) on MIPs and non-imprinted polymers (NIPs) were analyzed, showing higher adsorption capacity of MIPs due to the specific cavities. The modification with SDS or CTAB effectively eliminated non-specific adsorption in MIPs. The kinetic adsorption behavior further demonstrated the efficacy of MIP+--SDS/CTAB in the selective adsorption of SMX. Calibration curves showcase the methodology's analytical capabilities, achieving low limit of detection for SMX 6 ng mL−1 using MIP +-SDS. The stability study confirmed that the developed MIP +/--SDS/CTAB remains stable even at high temperatures, demonstrating its suitability for on-site applications. The methodology was successfully applied to detect SMX in milk and water samples, achieving promising recoveries. Overall, the electrostatic modification of MIPs with surfactants emerges as a valuable strategy for enhancing selectivity and performance in target molecule recognition and detection.

Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000ms, the device allows us to follow invitro reactions of biological molecules at resolution levels in the range of 3Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E.coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140ms.

Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink

BackgroundCellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink.ResultsWe used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs.ConclusionsIn an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.

Ultrasensitive Upconversion Nanoparticle Immunoassay for Human Serum Cardiac Troponin I Detection Achieved with Resonant Waveguide Grating.

Selective detection of biomarkers at low concentrations in blood is crucial for the clinical diagnosis of many diseases but remains challenging. In this work, we aimed to develop an ultrasensitive immunoassay that can detect biomarkers in serum with an attomolar limit of detection (LOD). We proposed a sandwich-type heterogeneous immunosensor in a 3 × 3 well array format by integrating a resonant waveguide grating (RWG) substrate with upconversion nanoparticles (UCNPs). UCNPs were used to label a target biomarker captured by capture antibody molecules immobilized on the surface of the RWG substrate, and the RWG substrate was used to enhance the upconversion luminescence (UCL) of UCNPs through excitation resonance. The LOD of the immunosensor was greatly reduced due to the increased UCL of UCNPs and the reduction of nonspecific adsorption of detection antibody-conjugated UCNPs on the RWG substrate surface by coating the RWG substrate surface with a carboxymethyl dextran layer. The immunosensor exhibited an extremely low LOD [0.24 fg/mL (9.1 aM)] and wide detection range (1 fg/mL to 100 pg/mL) in the detection of cardiac troponin I (cTnI). The cTnI concentrations in human serum samples collected at different times during cyclophosphamide, epirubicin, and 5-fluorouracil (CEF) chemotherapy in a breast cancer patient were measured by an immunosensor, and the results showed that the CEF chemotherapy did cause cardiotoxicity in the patient. Having a higher number of wells in such an array-based biosensor, the sensor can be developed as a high-throughput diagnostic tool for clinically important biomarkers.

Porous Laser-Scribed Graphene Electrodes Modified with Zwitterionic Moieties: A Strategy for Antibiofouling and Low-Impedance Interfaces.

Laser-scribed graphene electrodes (LSGEs) are promising platforms for the development of electrochemical biosensors for point-of-care settings and continuous monitoring and wearable applications. However, the frequent occurrence of biofouling drastically reduces the sensitivity and selectivity of these devices, hampering their sensing performance. Herein, we describe a versatile, low-impedance, and robust antibiofouling interface based on sulfobetaine-zwitterionic moieties. The interface induces the formation of a hydration layer and exerts electrostatic repulsion, protecting the electrode surface from the nonspecific adsorption of various biofouling agents. We demonstrate through electrochemical and microscopy techniques that the modified electrode exhibits outstanding antifouling properties, preserving more than 90% of the original signal after 24 h of exposure to bovine serum albumin protein, HeLa cells, and Escherichia coli bacteria. The promising performance of this antifouling strategy suggests that it is a viable option for prolonging the lifetime of LSGEs-based sensors when operating on complex biological systems.

A label-free fluorescent magnetic dual-aptasensor based on aptamer allosteric regulation of β-lactoglobulin

We presented a label-free fluorescent biosensor based on magnetic dual-aptamer allosteric regulation of β-lactoglobulin (β-LG) detection. The bovine serum albumin (BSA) acted as the bridge to connect amino-modified magnetic beads and aptamer, which synthesized pyramid-type probes (MBAP) with high capture and reduced nonspecific adsorption. Moreover, the original aptamer was tailored and then designed as a bivalent aptamer to fabricate allosteric signal probes (ASP). The ASP can both specifically capture β-LG and output the fluorescence signal. The detection mechanism is as follows. The combination of the dual-aptamer and β-LG triggered the allosteric change, resulting in the release of SYBR Green (SG I) from the allosteric signal probe and change signals. This method exhibits a broad linear detection range from 10 ng/mL to 1 mg/mL and the limit of detection reaches as low as 8.06 ng/mL. This study provides a highly generalizable strategy for protein biomolecular detection via replacing different target aptamers.

Challenges for Field-Effect-Transistor-Based Graphene Biosensors.

Owing to its outstanding physical properties, graphene has attracted attention as a promising biosensor material. Field-effect-transistor (FET)-based biosensors are particularly promising because of their high sensitivity that is achieved through the high carrier mobility of graphene. However, graphene-FET biosensors have not yet reached widespread practical applications owing to several problems. In this review, the authors focus on graphene-FET biosensors and discuss their advantages, the challenges to their development, and the solutions to the challenges. The problem of Debye screening, in which the surface charges of the detection target are shielded and undetectable, can be solved by using small-molecule receptors and their deformations and by using enzyme reaction products. To address the complexity of sample components and the detection mechanisms of graphene-FET biosensors, the authors outline measures against nonspecific adsorption and the remaining problems related to the detection mechanism itself. The authors also introduce a solution with which the molecular species that can reach the sensor surfaces are limited. Finally, the authors present multifaceted approaches to the sensor surfaces that provide much information to corroborate the results of electrical measurements. The measures and solutions introduced bring us closer to the practical realization of stable biosensors utilizing the superior characteristics of graphene.

Malathion Detection Using Molecularly-Imprinted Polymer Quartz Crystal Microbalance Sensor

The extensive use of pesticides can result in overexposure and soil, water, and produce residues. For instance, residues of malathion were found on some vegetables. Molecularly-imprinted polymers (MIP) have been recently developed for sensing of pesticide residues. This study prepared malathion-imprinted polymers via precipitation polymerization and deposited on quartz crystal microbalance (QCM) electrodes. FTIR spectroscopy proved the incorporation and removal of malathion in the matrix of MIP. SEM images revealed that MIP particles are larger than the non-imprinted polymer (NIP) particles due to the incorporation of malathion. Binding experiments were done using standard malathion solutions of 10 to 60 ppm. The MIP-QCM sensor had a greater response than the NIP-QCM sensor. This is due to the specific binding sites in the MIP matrix. On the other hand, the response of NIP-QCM sensor is attributed to the non-specific adsorption sites in its matrix. A sensitivity and detection limit of 1.62 Hz·L/mg and 5.67 ppm, respectively were determined for the MIP-QCM sensor. Lastly, the MIP-QCM sensor is stable and reusable up to three (3) cycles.

Wavelength-dependent photoelectrochemical response demonstrated by the determination of acetaminophen and rutin in differential molecularly imprinted polymers strategy

Herein, the excitation wavelength-dependent responses of the molecularly imprinted polymer (MIP) photoelectrochemical (PEC) sensors were investigated, using acetaminophen (AP), rutin (RT) and perfluorooctanoate (PFOA) as the model templates, pyrrole as functional monomer, CuInS2@ZnS/TiO2 NTs as the basic photoelectrode. With wavelength λ > 240 nm, the photocurrent of MIPPFOA enhanced at higher concentrations of PFOA. With increasing AP concentration, the photocurrents of MIPAP could decline with λ < 271 nm, not change at λ = 270 nm, or increase with λ > 270 nm. As RT concentration increased, the photocurrents of MIPRT could decrease (λ < 431 nm), not change (λ = 431 nm) or increase (λ > 431 nm). The PEC responses depend on the comprehensive interaction of two contrary mechanisms from the template molecules within the MIP membrane. The photocurrent is enhanced by the role of the electron donor for photo-generated holes but attenuated due to the steric hindrance effect and the excitation light intensity loss via absorption or scattering. The apparent molar absorption coefficient of AP and RT within MIP membranes are 9.1–19.4 folds of those measured from dilute solutions. By using a routine UV lamp as the light source, the photocurrents of MIPRT at 254 nm and MIPAP at 365 nm were used to determine RT and AP, with the detection limits of 5.3 and 16 nM, respectively. The interference from the non-specific adsorption of interferents on the surfaces of MIPAP and MIPRT was reduced by one order of magnitude via a differential strategy.

An antifouling electrochemical aptasensor based on a polydopamine-polyzwitterion copolymer for tetracycline analysis

Matrix interference resulting from the nonspecific adsorption of non-target components, particularly proteins (fouling), onto sensor surfaces poses a persistent challenge in electrochemical detection of food hazards. The development of antifouling sensor surfaces presents a viable approach to mitigate nonspecific adsorption. In this study, a novel antifouling electrochemical aptasensor, utilizing a zwitterionic polymer, was developed for the sensitive, accurate, and selective detection of tetracycline (TC) in milk. This sensor employs a poly (dopamine)-poly (sulfobetaine methacrylate) (PDA-PSBMA) antifouling copolymer, which is synthesized through an in-situ initiated copolymerization of dopamine on the sensor's surface. Subsequently, the thiol-containing aptamers were immobilized onto the PDA-PSBMA coating through a Michael addition reaction with the poly(dopamine). The resulting antifouling electrochemical aptasensor exhibited robust antifouling performance in various single protein solutions and diluted milk samples, coupled with sensitive and selective recognition of TC. The sensor demonstrated a broad linear response range of 0.1–1000.0 ng mL−1 and a low limit of detection (LOD) of 68.0 pg mL−1. The antifouling electrochemical aptasensor proved effective in assaying TC in diluted milk, with recoveries ranging from 100.0 % to 104.4 %, eliminating the need for additional pretreatments due to its exceptional resistance to nonspecific adhesion.

Label-free detection of biomolecules using inductively coupled plasma mass spectrometry (ICP-MS).

Bioassays using inductively coupled plasma mass spectrometry (ICP-MS) have gained increasing attention because of the high sensitivity of ICP-MS and the various strategies of labeling biomolecules with detectable metal tags. The classic strategy to tag the target biomolecules is through direct antibody-antigen interaction and DNA hybridization, and requires the separation of the bound from the unbound tags. Label-free ICP-MS techniques for biomolecular assays do not require direct labeling: they generate detectable metal ions indirectly from specific biomolecular reactions, such as enzymatic cleavage. Here, we highlight the development of three main strategies of label-free ICP-MS assays for biomolecules: (1) enzymatic cleavage of metal-labeled substrates, (2) release of immobilized metal ions from the DNA backbone, and (3) nucleic acid amplification-assisted aggregation and release of metal tags to achieve amplified detection. We briefly describe the fundamental basis of these label-free ICP-MS assays and discuss the benefits and drawbacks of various designs. Future research is needed to reduce non-specific adsorption and minimize background and interference. Analytical innovations are also required to confront challenges faced by in vivo applications.

Sensitivity-improved blocking agent-free fluorescence polarization assay through surface modification using polyethylene glycol.

Fluorescence polarization (FP) assays are widely used to quantify biomolecules, and their combination with microfluidic devices has the potential for application in onsite analysis. However, the hydrophobic surface of polydimethylsiloxane (PDMS)-based microfluidic devices and the amphiphilicity of the blocking agents can cause the nonspecific adsorption of biomolecules, which in turn reduces the sensitivity of the FP assay. To address this, we demonstrated an FP assay with improved sensitivity in microfluidic devices using a polyethylene glycol-based surface modification to avoid the use of blocking agents. We evaluated the effectiveness of the modification in inhibiting nonspecific protein adsorption and demonstrated the improved sensitivity of the FP immunoassay (FPIA). Our study addressed the lack of sensitivity of FP assays in microfluidic devices, particularly for the quantification of low-abundance analytes.

Interaction of Pseudomonas aeruginosa with surface-modified silica studied by ultra-high frequency acoustic wave biosensor

Aim: This study aimed to examine the amount of surface non-specific adsorption, or fouling, observed by Pseudomonas aeruginosa (P. aeruginosa) on a quartz crystal based acoustic wave biosensor under different flow conditions with and without an anti-fouling layer. Methods: An electromagnetic piezoelectric acoustic sensor (EMPAS) based on electrode free quartz crystals was used to perform the analysis. Phosphate buffered saline (PBS) was flowed over the crystal surface at various flow rates from 50 μL/min to 200 μL/min, with measurements being taken at the 43rd harmonic (~864 MHz). The crystal was either unmodified, or modified with a monoethylene glycol [2-(3-silylpropyloxy)-hydroxy-ethyl (MEG-OH)] anti-fouling layer. Overnight culture of P. aeruginosa PAO1 (PAO1) in lysogeny broth (LB) was injected into the system, and flow maintained for 30 min. Results: The frequency change of the EMPAS crystal after injection of bacteria into the system was found to change based on the flow rate of buffer, suggesting the flow rate has a strong effect on the level of non-specific adsorption. The MEG-OH layer drastically reduced the level of fouling observed under all flow conditions, as well as reduced the amount of variation between experiments. Flow rates of 150 μL/min or higher were found to best reduce the level of fouling observed as well as experimental variation. Conclusions: The MEG-OH anti-fouling layer is important for accurate and reproducible biosensing measurements due to the reduced fouling and variation during experiments. Additionally, a flow rate of 150 μL/min may prove better for measurement compared to the current standard of 50 μL/min for this type of instrument.